Detective DNA Parwinder Chawla 1998

DETECTIVE DNA
CSIR GOLDEN JUBILEE SERIES
PARVINDER CHAWLA
DETECTIVE DNA
PARVINDER CHAWLA
National Institute of Science Communication Dr K. S. Krishnan Marg New Delhi 110012 India.
Detective DNA Parvinder Chawla

1998, National Institute of Science Communication (CSIR) First Edition: January 1998 ISBN: 81-7236-175-0
CSIR Golden Jubilee Series Publication No. 21 Series Editor Volume Editor Cover Design Illustrations Production Printing Dr Bal Phondke S.K. Nag Pradip Banerjee Sushila Vohra, Neeni Vijan, Malkhan Singh, Yogesh Kumar, J.M.L. Luthra, Harjeet Singh Kaushal Kishore, Shiv Kumar Marhkan, Rohini Kaul, Pamela Khanna, Vijay Sharma Gopal C. Porel, Sudhir C. Mamgain, Rattan Lai, Tika Ram, Om Pal, Jawahar Lai
Designed, Printed and Published by National Institute of Science Communication (CSIR) Dr K.S. Krishnan Marg, New Delhi 110012
Foreword
The Council of Scientific & Industrial Research (CSIR), established in 1942, is committed to the advancement of scientific knowledge, and economic and industrial development of the country. Over the years CSIRhas created a base for scientific capability and excellence spanning a wide spectrum of areas enabling it to carry out research and development as well as provide national standards, testing and certification facilities. It has also been training researchers, popularizing science and helping in the inculcation of scientific temper in the country. The CSIR today is a well knit and action oriented network of 41 laboratories spread throughout the country with activities ranging from molecular biology to mining, medicinal plants to mechanical engineering, mathematical modelling to metrology, chemicals to coal and so on. While discharging its mandate, CSIR has not lost sight of the necessity to remain at the cutting edge of science in order to be in a position to acquire and generate expertise in frontier areas of technology. CSIR's contributions to high-tech and emerging areas of science and technology are recognised among others for precocious flowering of tissue cultured bamboo, DNA finger-printing, development of non-noble metal zeolite catalysts, mining of polymetallic nodules from the Indian Ocean bed, building an all-composite light research aircraft, high temperature superconductivity, to mention only a few. Being acutely aware that the pace of scientific and technological development cannot be maintained without a steady influx of bright young.scientists, CSIR has undertaken a vigorous programme of human resource development which includes, inter alia, collaborative efforts with the University Grants Commission aimed at nurturing the budding careers of fresh science and technology graduates. However, all these would not yield the desired results in the absence of an atmosphere appreciative of advances in science
and technology. If the people at large remain in awe of science and consider it as something which is far removed from their realms, scientific culture cannot take root. CSIR has been alive to this problem and has been active in taking science to the people, particularly through the print medium. It has an active programme aimed at popularization of science, its concepts, achievements and utility, by bringing it to the doorsteps of the masses through both print and electronic media. This is expected to serve a dual purpose. First, it would create awareness and interest among the intelligent layman and, secondly, it would help youngsters at the point of choosing an academic career in getting a broad-based knowledge about science in general and its frontier areas in particular. Such familiarity would not only kindle in them deep and abiding interest in matters scientific but would also be instrumental in helping them to choose the scientific or technological education that is best suited to them according to their own interests and aptitudes. There would be no groping in the dark for them. However, this is one field where enough is never enough. This was the driving consideration when it was decided to bring out in this 50th anniversary year of CSIR a series of p r o f u s e l y illustrated and specially written popular monographs on a judicious mix of scientific and technological subjects varying from the outer space to the inner space. Some of the important subjects covered are astronomy, meteorology, oceanography, new materials, immunology and biotechnology. It is hoped that this series of monographs would be able to whet the varied appetites of a wide cross-section of the target readership and spur them on to gathering further knowledge on the subjects of their choice and liking. An exciting sojourn through the wonderland of science, we hope, awaits the reader. We can only wish him Bon voyage and say, happy hunting.
Preface
The book aims to offer the non-specialist reader an insight into the exciting technology of DNA fingerprinting which encompasses the use of many sophisticated biotechniques. It is, therefore, an earnest attempt to make the understanding of the biotechniques, involved in profiling DNA, accessible to a broader audience. The running theme of this book shows how a single stranded DNA segment tagged with a radioactive molecule, works like a detective. For, it triumphantly searches out its complementary DNA sequence from among several others a feat as daring as finding a needle in a haystack. Still exciting is how this molecular union, an invisible event, is translated into a visible form. The hallmark of DNA profiling is its power to reveal, with utmost 'precision', the unique molecular signature that each one of us carry in our genes. Thanks to specially designed detectives which come to the rescue in parentage disputes and even solve forensic cases. Extending this technology to all living organisms, specific detectives can help identify each one of them. Based on revealing certain specific or unique stretches of DNA in an organism, DNA profiling has brought about a revolution in the diagnosis of infectious diseases besides its plethora of uses in food industry and agriculture. Predictive testing of genetic disorders by detecting faulty genes is also possible. It is to share with laymen the knowledge on this far-reaching technology and its tremendous potential that I have written this volume. Readers reactions are welcome.
Acknowledgements
Writing a simple and easy-to-understand book on DNA fingerprinting was a challenging task but, to be frank, the experience was equally exciting and enjoyable. I thank Dr G.P. Phondke for giving me this opportunity. After approving the book's'outline, he at once gave me the green signal for going ahead. Charged with the excitement of writing my first popular science book, I prepared the first draft. I am grateful to Shri S.K. Nag for patiently editing the manuscript and providing constant support. My husband, Anil, deserves the credit for critically reading the text and giving valuable suggestions in spite of his busy schedule. His immense confidence in me truly qualifies him for my special thanks. I am thankful to Ms Vijay Sharma for keying in the handwritten draft and taking countless corrections. Shri Shiv K. Markhan and Ms Rohini Kaul deserve my thanks for formatting the final pages. I am grateful to Dr Indira Nath, Dr P. Usha Sarma, Dr Jaya Tyagi and Dr Sher Ali for providing necessary inputs about their research work. Dr Jaya Tyagi's help in taking some photographs is acknowledged. It was very nice of Dr Lalji Singh to provide interesting results of DNA analysis which helped solve some paternity disputes. I am indebted to Shri Pradip Banerjee and his highly competent team of artists for making excellent illustrations which both enrich and complement the text. The sincere efforts of Shri Banerjee's team has indeed given the book, a magical touch, for which I am extremely grateful. Finally, this book could see the light of the day due to the hard labour put in by Shri K.B. Nagpal and his talented colleagues in the printing section. To them all, I would like to express my appreciation and gratitude.
Dedicated to
my dear parents Late Shri P.S. Sindhoo and Smt. Kulwant Kaur To whom I owe my existence and above all, my genetic blueprint Cheena
Contents
The Ultimate Identity The Power of Repeats Gifting the Torch The Masterplan From One to Many Detecting Defective Genes Catching the Culprit: A microbe A Boon to Food Industry Hurdles to Hi-Tech Glossary
...
. . . 16 ... 34
...46 ...66 ...80 . . . 104 . . . 118 . . . 126 . . . 133
ang! Bang! Bang! Three shots rang up in quick succession. Two bullets entered the victim's head and the third missed and hit the wall. Death was instantaneous. The body was dumped in a car and taken to a restaurant where it was consigned to the hot flames of a tandoor. However, to make the disposal convenient, the limbs had been brutally severed.
The Ultimate Identity
Seems to be a clip from a horror story? Well, it is a real-life happening that took place in this country. Yes, the victim of this horrifying, spine-chilling murder was a young woman. The half charred body was later recovered. But to add to the tragedy no one, not even her parents and relatives came forward to claim the body on the pretext that it was burned beyond recognition. Then how was it concluded that the body was really of that woman? The murder mystery began to unfold w h e n the police broke open the house where the murder occurred and found many clues an empty cartridge, some letters and most importantly, blood stains on the mattress and carpet. T h e p a t c h of c a r p e t c a r r y i n g blood stains and the bone samples taken from the unidentifiable
DETECTIVE DNA
body along with blood samples of the woman's parents were sent to the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, a constituent national laboratory of the Council of Scientific & Industrial Research, for conducting a special and highly sophisticated test whose results, if positive, would be a clinching evidence to the victim's identity. The,test proved positive as the results showed a clear match between the victim's body tissues and blood of her parents. So, the leaping flames of the tandoor carrying with it the stench of burning human flesh did not carry away with them the victim's identity, thanks to the modern hi-tech methods which revealed the fact from the dried blood stains and bone sample of the murdered woman. Well, what can a person's blood samples reveal? Normally, the blood obtained from the site of crime is tested for its blood group and matched with those of the victim and the suspects. Blood can be classified under different blood group systems, the most common being the ABO system which was discovered in 1900 by Karl LANDSTEINER. This is based on the fact that the red cells of blood in every individual carry on their surface a specific molecular entity, the antigen. According to the ABO system, human blood can be differentiated into four groups; A, B, AB and O which is based on the presence or absence of the two antigens called A and B. The AB group has both antigens A and B on the surface of red cells. A and B groups have antigens A and B respectively, whereas in O group both these antigens are missing. The blood plasma of these four types of blood groups contains special antibodies. Type A blood has anti-B antibodies which specifically bind to antigen B and plasma of type B blood contains anti-A antibodies which similarly bind to antigen A. This antigen-antibody binding results in clumping of red cells. Blood type O has both these antibodies while AB blood group has none. Cross matching of the donor's blood type with that of the recipient's before a blood transfusion is, therefore, necessary. Similarly, blood can be tested for the M
THE ULTIMATE IDENTITY
Blood groups
The ABO system of matching blood groups
and N antigens and the Rh antigen besides a few other blood group systems. From bloodstains, one can even identify the sex of a particular person. Unlike the red cells, the white cells present in blood contain a nucleus in which lies the chromosomes the carriers of heredity. Microscopic study of chromosomes reveals the presence of two big X-chromosomes in females
DETECTIVE DNA
whereas in males there is just one X-chromosome and a tiny Y-chromosome. The two X-chromosomes actually appear as a 'drumstick' inside the nucleus of cells in females, which is not present in males. The blood stains may even be tested for certain special enzymes and proteins found in some human
Drumstick
White blood cells
The drumstick is unique to women
races and populations. Some of these even display different structures in different individuals, although their basic functions remain unaltered. Any resemblance in their structures in two individuals could be an evidence for them being related. Notwithstanding the possible relatedness revealed between two individuals, the results of these tests cannot stand as 'foolproof' evidence to one's identity Positive identification of a person using these techniques is not 100% even if several specific characteristics of blood are tested in conjunction. The million-dollar question, that is whether the blood is of the victim or any of the suspects, can in no way be conclusively answered by these methods. It seems, as if, the entire exercise of identifying a person from blood stains has gone in vain. More so, if the blood samples reach late for testing, the proteins and enzymes are degraded and the tests become invalid. Nevertheless, with the advent of sophisticated tech-
niques of biotechnology, many untouched pastures have been explored. To rescue came the miraculous substance, the master molecule of life called deoxyribose nucleic acid (DNA). The basis of all life forms we see, it is this wonderful molecule which constitutes the 'blueprint' of every living organism and by which traits are passed on from one generation to another. It is now essentially clear that it is the DNA which differentiates every living species from any other. This coded genetic information present in every cell of the body of a person can be profiled to produce the most authentic identity card of any biological specimen. The sophisticated technology that facilitates the identification of individuals at the genetic level is popularly known as 'DNA fingerprinting', although it is more appropriate to call it 'DNA profiling'. This technology is the brain child of a British geneticist, Alec JEFFREYS. Working at the U n i v e r s i t y of Leicester, Jeffreys laid its foundation in 1984. Based on his findings, any individual can be specifically identified on the basis of his or her unique pattern of DNA obtained using special techniques. This genetic analysis is actu- Alec Jeffreys the genius who laid the ally based on identifyfoundation of DNA profiling technology ing tiny segments of the
DETECTIVE DNA
hereditary material which testify the unique 'molecular signature' that every person on this planet is endowed with and which cannot be altered in his/her lifetime. So, each one of us can be differentiated on the basis of our genetic material which, although similar in most respects as we are all members of the human species, has distinctive regions. So, the DNA profile of each of five billion human beings differs with, of course, one exception identical twins. The genetic material of identical twins is an exact copy of each other as their life begins from the same entity, the zygote which is the product of the union of two hereditary cells, the mother's egg and the father's sperm. The zygote breaks into two halves and each half divides and grows into an individual. Thus, the genetic identity cards of identical twins would also be identical. To obtain a DNA profile, the basic requirement is the availability of any body tissue. From the cells of these tissues the genetic material could be extracted. This biological sample can be anything including a. blood stain, a piece of hair with its root, few drops of semen, skin cells, a mouth swab, cells of the bone marrow or any other tissue. At the heart of the DNA profiling technology is a tiny but specific segment of DNA which can be likened to a 'smart' detective. The ingenuity of this detective lies in probing untiringly the six-feet long thread of the genetic material, contained in every cell of a person, for certain chosen segments. The 'bar code' pattern of these segments ultimately defines the identity of that person at genetic level. This bar code appearance of the genetic signature is so because the final result is visualized as an array of light and dark bands of varying thickness present one below the other with unequal separations between adjacent bands. It is this which differs from individual to individual. It was the modern day Sherlock Holmes a detective par excellence albeit a molecular entity that was ultimately em-
Blood cells
Hair
Check cells
Blood stain
Mouth swab
Skin tissue Bone marrow cells
_ Spermatozoa/
sperm
A DNA profile can be made from any available body tissue of a person
ployed to reveal the identity of the woman who was shot and barbecued. Such cases where the dead body is unidentifiable on account of being mutilated, burned or decomposed are now increasingly seeking the help of DNA profiling technology. The dead person's DNA profile is compared with those of putative parents, siblings or relatives. Identity crises can, therefore, be easily overcome by pressing into service these specially designed detectives. The technology of DNA profiling has come a long way since the inception of 'fingerprinting' per se. A common
DETECTIVE DNA
thread that links the technology of DNA profiling with fingerprinting is that both are based on matching certain patterns. Be it the pattern of fingerprints or the genetic pattern, both are unique to an individual. So, the term 'DNA fingerprinting/ as the technique is referred to in common parlance, has actually been derived from its conventional counterpart. The science of fingerprinting is based on the fact that every individual has on the skin of the underside of hands and feet fine raised lines which form a set pattern of tiny conical
Loop Arch Whorl
Composite
The four standard fingerprint patterns
elevations. Especially, the fingertips have quite distinctive raised lines. The pattern formed by skin ridges may be an arch, a loop, a whorl or a composite. Those patterns further have features, like forks, lakes, spurs and islands. When a person touches an object, the imprints of these patterns are left behind on the surface in contact. Interestingly, all individuals including identical twins have different fingerprints which remain unchanged for lifetime. This is seen from individual fingerprints obtained on pressing the finger ends and thumbs on an inked pad which are then
pressed on a piece of paper for obtaining the impression of patterns in the skin. Lifting faint fingermarks from different surfaces and interpreting them is the hallmark of fingerprinting technology. Several techniques are employed to lift fingermarks. Once the fingermarks have been lifted, matching these with inked fingerprints is done. This requires an intense concentration besides good eyesight. What a fingerprint expert looks for is the matching patterns and features between the two. For positive identification, several minute details of the fingermarks should match those of the fingerprint. The matching is laborious as it is done manually. Of the m a n y technological developments which have taken place in forensic science for over a century, the most revolutionary one, however, has been the profiling of DNA. Classic fingerprinting, analyzes the phenotypic traits which indicate the external appearance of an individual. These, in fact, in many cases could be shared in different person. So, in the strictest sense, classic fingerprinting tests lack the ability of identifying a person conclusively, although these techniques have been popular in the past. On the other hand, D N A analysis directly provides genotypic information which determines the genetic make-up of an organism. It was the year 1986 that witnessed a watershed forensic triumph. The first mass genetic fingerprinting was done in Leicestershire countryside where two young teenage girls had been raped and murdered. Alec Jeffreys and his colleagues took up the gigantic task of screening the blood and saliva samples of 5500 men who lived in the area where the crime occurred. The culprit identified at last was, Colin Pitchfork, a 27-year old baker the first criminal in history to be convicted on the basis of DNA profiling. This also freed an innocent suspect. This incidence was a stepping stone in proving the tremendous potential of the new DNA technology. Not surprisingly, it is today, the most sophisticated tool
10
DETECTIVE DNA
in the repertoire of a forensic expert. Thousands of criminal cases including rape and murder, both in USA and Britain have been concluded logically since the advent of DNA profiling technology. Databases of the genetic fingerprints of criminals have been set up in many parts of USA and Britain. The world's first National DNA database has been set up at the Britain's Forensic Science Service (FSS) in Birmingham. It aims to facilitate the systematic collection of tissue samples, followed by analysis and storage of DNA profiles from suspects. Eventually, about five million records would be held on the UK database. Unlike in forensic science, where DNA profile of two completely unknown individuals is compared for a match which may be repeated as many times the number of suspects, solving cases of disputed parentage is simpler and less messier. It is so, because parts of the genetic signature of a child come from the mother and the remaining find a perfect match with the DNA profile of the father. A child's parentage can, therefore, be authenticated beyond any doubt, by simply comparing the DNA profile of the child with that of its putative parents. The molecular detective plays 'Solomon the wise' in such cases. Most of us are familiar with this interesting story of King Solomon who very cleverly solved the dispute between two women both claiming a child to be theirs. The actual mother unable to bear the king's order of cutting the^child into two halves, at once stepped down from her claim. The child was of this woman, so decided the wise King. Such is the wisdom of the modern-day detective in settling parental disputes. Thousands of such cases all over the world have been solved by bringing to trial the genetic material of putative parents. In India as well many cases of such disputed parentage have been solved. In one case, a 13-month old child was in
THE ULTIMATE IDENTITY 21
W h o is the actual mother? King Solomon finds out
12
DETECTIVE DNA
Alleged Mother 1
Alleged Mother 2
Father
Child
the news. Claiming to be its biological mother, a young woman launched a legal battle against a man who took away the baby within two days of its birth. This man and his legally wedded wife, claimed the baby to be theirs. However, hospital medical records showed that the woman who registered this case was the real mother. To resolve this tricky dispute, the Supreme Court took resort to DNA profiling technology
13
and ordered these tests to be conducted for the couple, petitioner and the child. As D N A analysis provides an unambiguous and conclusive proof of parent-child relationship, these tests are helping to reunite children with their actual parents. Families whose members were separated by natural disasters, war or other violence have been reunited using these new genetic tests. In Argentina, for example, many people were abducted during the military rule (1976-1983). These people presumably died as they never returned. However, according to an Argentinian human rights group, the Grandmother's of Plaza de Mayo, many lost children are believed to be still alive as
DNA profiling unites lost children with their grandparents and relatives
14
DETECTIVE DNA
they were adopted by military personnel involved in the murder of the child's natural parents. A genetic data bank storing the blood samples of grandparents and relatives of these lost children was established in May 1987. By matching the DNA profile of one or more grandparents with that of a lost child's, about 40 such missing children have been already reunited with their families. The genetic tests here are based on the fact that a child inherits a quarter of its genetic material from each grandparent. The molecular detective looks for these inherited sequences in the child's genetic material. The infallibility of DNA profiling technology in establishing a child's parentage has been invaluable in solving many immigration disputes as well. In 1985, it helped examining 36 families who wished entry to Britain where their relatives were staying, thus providing immigration authorities a clear evidence of relationships between different individuals of a family. Realizing the potential of this technology, the Armed Forces Institute of Pathology, Gaithersberg, USA, has undertaken a massive collection of DNA samples from every member of the armed services. These samples are stored in freezers in DNA specimen repositories. Each sample comprises blood blots on cards, sealed in individual envelopes. For the test, a tiny portion of the blotted blood is punched out from which blood cells are isolated. A computer database of all samples has been prepared. The first test from these preserved samples was done to identify a soldier burnt to death in a car accident. Later, a pilot killed in a plane crash was also identified. Many repositories of this kind are poised to come lip as the technology gains wider grounds. A vast spectrum of uses of DNA profiling have been realized in different fields owing to the essence of the technology that genetic material of any living organism, be it a plant, an animal or a human being, can be profiled exposing those
15
Broad \ Applications of DNA Profiling y \
segments of its blueprint which are unique to it and hence confirm its identity. Among others, it includes the specific diagnosis of various genetic and infectious diseases in man which is poised to bring a revolution in medical diagnostics. Besides, it helps in ascertaining the pedigree of an animal and identifying seed stocks of plants. Numerous uses of the technology have been realized in the food industry as well.
s>
The Power of Repeats
ortyfour years ago there occurred a scientific breakthrough whose impact dramatically changed the future course of science. The historical day was April 2, 1953 when a young American biologist James D. WATSON and an English Physicist Francis H.CRICK proposed a model of D N A structure. With the elucidation of the shape of DNA, an explosion of new developments followed in many areas of genetics and biochemistry. An exhaustive study of this celebrated molecule led to one scientific triumph after another. Today, most of us are familiar with the structure of this biologically significant molecule. As proposed by Watson and Crick, the DNA molecule has a structure like that of a sleek spiral staircase. Not only is it thin but a long one stretching up to roughly six feet if uncoiled. It is tightly packaged in 46 chromosomes present inside each of the trillions of cells of our body. The bannisters of this staircase comprise p h o s p h a t e units and simple sugar molecules of the kind known as deoxyribose in alternating order. These molecules present on the outside of the long DNA chain constitute its
THE POWER OF REPEATS
17
James D. Watson (top) and Francis H. Crick (centre) revealed the structure of DNA a ground breaking work for which they won the Nobel Prize along with Maurice Wilkins (bottom)
18
DETECTIVE DNA
backbone, thus performing just a structural role. The steps of the spiral ladder consist of two loosely linked organic molecules called bases. So, structurally the D N A molecule is not as complicated as are most organic molecules. Its simple basic unit is called a nucleotide that consists of a phosphate, a sugar and a nitrogenous base. However, the molecule has a spiral structure as its two bannister-like strings interwound to form a helical structure. That is why D N A molecule is popularly called the 'double helix'. The entire genetic message that controls the chemistry of every cell of the body instructing it to act in a specific way is actually written in the language of just four nitrogenous bases. There are two classes of bases in DNA the purines and the pyrimidines. These letters are adenine, guanine, cytosine and thymine. The novel feature of the double helix is the manner in which only specific letters of the two opposite polynucleotide chains are held together by hydrogen bonds. The base adenine (purine) always pairs with thymine (pyrimidine) and guanine (purine) with cytosine (pyrimidine). This binding is so specific that respective base pairs fit together like prongs into sockets. This means that the two DNA strings in a double helix are 'complementary' and not identical. This four letter code reads in a line along the DNA. However, it is the specific combination of these bases forming a unique sequence that spells out the coded message in the D N A molecule. Just as the 26 alphabets of English language can be assembled in millions of ways forming as many meaningful sentences as would make up the entire past, present and even future literature, so are these four letters of the genetic code arranged in almost unlimited number of unique sequences, specifying the genetic information for the entire living world. A defined sequence of the four bases constitutes a 'gene' which may be a few or several hundred base pairs long. Genes are the storehouses of the blueprints of
THE POWER OF REPEATS Base pair
19
Thymine
Adenine
Cutosine
Guanine
Double helix
Nitrogenous base Phosphate (P) i Deoxyribose sugar (S)
Nucleotide
One nucleotide
T h e structure of the d o u b l e helix. C o m p l e m e n t a r y b a s e s bind to e a c h o t h e r just as p r o n g s fit into sockets (inset)
20
DETECTIVE DNA
molecules called proteins the key biochemicals that control life's infinite variety. It is known that 99 per cent of base sequence is same in the D N A of all human beings. Only the sequence of very short stretches of DNA, sprinkled over the total D N A of a cell
Non coding -segments
* Chromosome
txon Intron
A genetic stammer? Unique and repeating bits of DNA are scattered all over the total chromosomal DNA
Dispersed repeats .
"~~Tandetnly repeated sequences
Exun
21
which is about three billion base pairs, differs from person to person. Hi-tech methods involved in DNA profiling enable to hunt with unmatched specificity these variable unique sequence patterns or 'fingerprints' resting inside the DNA of every person. Of the total DNA, about one in 1000 base pairs is a site of variation in the population. Understandably, for matching the DNA of two persons one cannot scan the entire length of their respective DNA molecules. All one looks for are the sequences unique to that person. Now, where are these sequences exactly present in the human DNA? Alec Jeffreys provided an answer to this question although he happened to set his eyes on such variable segments of DNA by chance. It all began in the year 1984 when he was studying the gene for myoglobin a protein that stores oxygen in the muscles. What the inquisitive young scientist observed was that part of this gene did not carry any instructions for the manufacture of myoglobin. All that these segments of DNA contained were the unusual sequences of bases which were repeated several times a kind of a genetic stammer. They were seemingly harmless. Now whether these repeating bits of DNA could be of any use, thought Jeffreys. His conclusion was 'yes'. He believed that these variable repeating segments, about 10-15 base pairs long, could act as genetic markers for the myoglobin gene which would help in tracking down the location of this gene on a particular chromosome. He went on to isolate these segments and then introduced them into bacteria. As the latter multiplied, so did the introduced segments of DNA forming a clone. This technique is called cloning. Jeffreys extracted these segments from bacteria and labelled them with a radioactive chemical. In essence, the first 'detective' DNA was born. Owing to the complementary nature of DNA, it was clear that binding of this labelled DNA to a DNA sample would reveal the presence of the repeat sequences in the myoglobin gene. Jeffreys further developed methods by which the binding of the detective DNA with its target se-
22
DETECTIVE DNA Gene for antibiotic gene resistance
foi^S"
Foreign
gene
Uptake by bacterial
cell
C l o n i n g a foreign g e n e in b a c t e r i a
quertces could be translated into a visual record on an X-ray film. This picture having a bar code appearance was seen to be individual specific. The story does not end here. Jeffreys found that repetitive DNA was not just in the myoglobin gene but it was at innumerable sites in the entire genetic material of a person. More exciting was that the pattern of repeats spread over the blueprint differed from person to person. This was because the length of the repeat sequence, the number of times it is repeated and its exact location in the long DNA chain were all unique to every individual. Most genes like myoglobin have such interspersing noncoding regions called 'introns' or simply junk DNA. The segments of repetitive DNA present in these regions are called 'minisatellites' as like a satellite they are around a
23
Stylus
Record
grooves'
Turntable
Tone arm Pick up
Speed control
Just as a phonograph needle when stuck in a groove produces a stammering note, certain stretches of DNA are repeated a variable number of times in human DNA. Close-ups of stylus and record grooves (inset)
particular universally shared gene. Interestingly, the coding regions of human DNA that house about one lakh genes containing recipes of all the proteins that body requires, actually constitutes just five per cent of the total DNA. The remaining 95 per cent of the DNA comprises the non-coding junk DNA. The latter may be either interspersing a gene or
24
DETECTIVE DNA
present between two adjacent genes. The function of the highly redundant DNA segments lying y silent' or inactive in the junk DNA is still unknown. To understand it better, consider the sequence of base pairs in a DNA molecule to be like a phonograph record. As long as the needle moves in a circular groove, one hears a meaningful tune. But it is a common experience that at times the needle gets stuck in a groove with the outcome that the same notes are repeated over and over again before the needle is placed properly correcting the error. These notes of the phonograph record that sound like a stammer may irritate a listener but the silent repeats sitting inside the DNA molecule play a very 'powerful' role in proving one's identity. The sequence of DNA repeated a variable number of times is called the 'core' sequence. A person may have several thousand repeats of a core sequence or may have it occurring only once. In scientific jargon, these regions are termed 'variable number of tandem repeats' (VNTRs). They were first studied and described as 'hypervariable' segments of DNA in 1980 by Arlene WYMAN and Ray WHITE of the University of Utah. However, it was Jeffreys who first used them for forensic identification as he found a 'trick' to count these repeats. By the sophisticated techniques involved in DNA analysis one can reliably identify several variable or 'polymorphic' regions in a DNA sample, ultimately proving that a particular combination of these segments in a molecular signature is unlikely to be found in anybody else. This forensic testing basically involves the preparation of an enzyme-cut D N A profile of an individual. The special enzyme, a molecular scissors used is called restriction endonuclease which recognizes only a particular base sequence. Several such enzymes are known today. The VNTRs that are useful for forensic analysis are those which happen to be flanked by pairs of sites that a particular restriction enzyme recognizes and can cleave. These sites where the enzyme acts to cut the D N A molecule are present far apart if the VNTR is
25
Length (in Kilobases)
DNA profiles of 11 individuals show that a region of DNA varies in all of them. Each individual has inherited one fragment from the father and one from the mother. Ray White at work
a long one and the same will be closely located if not many copies of that segment are present in that VNTR. The enzyme, however, does not cleave within the repeats. Special techniques then help to sort out these cut segments on the basis of their size. The length of the segment is dictated by the number of repeats the VNTR contains. It is this difference in length of a VNTR in different persons that is the key to
I.-.
K)
ClJ
26
DETECTIVE DNA
D N A profiling. Hence, the scheme developed by Jeffreys for measuring the lengths of fragments of D N A containing the repeat sequences is also technically known as 'Restriction Fragment length Polymorphism' (RFLP) testing. For obtaining the visual bar code pattern of a DNA profile or the RFLP pattern which is actually the picture of sizesorted VNTR fragments, a detective D N A referred to as a 'probe' is pressed into service. This detective, tagged with a radioisotope may have 10 to thousands of nucleotides which are complementary to a highly variable repeat sequence. It may be prepared by cloning or synthetic methods. This specially designed probe binds to that particular V N T R segment, both long and short ones, from among several unmatching or non-complementary stretches of DNA. This otherwise impossible task is seemingly "elementary" for the detective. The final result or the DNA profile showing the varying lengths of VNTRs, that is, the lengths of which vary in different individuals, is obtained on a X-ray film. The detective D N A which probes a D N A sample for specific sequences can be of two types: Single-locus probe and multi-locus probe. A single-locus detective binds to such VNTRs or core sequences which occur at only one locus or site in the total genetic material. The chromosomal location of such VNTRs is well defined. The D N A profile obtained using such probes consists of only two bands as in every individual there are two copies of a particular V N T R one from the mother and the other from the father. However, if that VNTR is of the same length in both the parents of a person then the latter's DNA profile will show a single band and he/she would be a 'homozygote' for that D N A sequence. A 'heterozygote', on the other hand, inherits two copies of a V N T R that is, one from each parent, which are of different lengths. Hence two discrete bands appear in the person's D N A profile. The precise length of bands in a D N A profile is calculated by measuring their position relative to the profile
THE POWER OF REPEATS Maternal line Grandmother Grandfather Paternal line Grandmother Grandfather
27
Children
Pattern of inheritance of a variable stretch of D N A over three generations
28
DETECTIVE DNA
of a " m a r k e r " D N A having DNA fragments of known size. This is done as simple visual inspection could be deceptive. Say a core sequence, namely CAGA is repeated five times at a specific locus in a person's DNA, 50 times in his father's D N A and 500 times, at the same site, in his uncle's. DNA profile using a single-locus probe for that V N T R can identify each of-these individuals. Similarly, another V N T R at some other locus may help identify different persons, related or unrelated. Nevertheless, a match by sheer chance in two individuals cannot be ruled out if only one locus is tested. That is, it is possible, in rare cases, that two persons just happen to have a V N T R of the same length. This problem can, however, be overcome by using a 'cocktail' of several single-locus probes. As two or more loci are tested, the chance of a coincidental match of different VNTRs present at well defined loci, becomes vanishingly small. Normally, in forensic identification, three or four different single-locus probes are used together so that the D N A profile produced has six or eight bands respectively. This understandably improves the validity of the test. Single-locus probes are mostly used for identifying a mixture of biological samples. For example, if a vaginal swab from a rape victim yields six bands using a single-locus probe it can be concluded that it is a case of multiple rape. Using single-locus probes a large number of samples can be screened. These probes can produce a clear D N A pattern even if sample D N A is present in little quantity or is partially decomposed. This is because the length of these probes is usually long and hence give a strong signal on binding to complementary regions in sample DNA. On the other hand, a multi-locus probe binds to repetitive regions or loci that occur at thousands of sites in the total human DNA. The DNA pattern obtained using a single multi-locus probe has a typical bar code appearance having about 20 or more interpretable bands. One half of these bands are inherited from the mother and one half is from the
29
Consecutively repeated segment (VNTR)
mm
Like a smart detective, a DNA probe reveals the identity of different persons as a stretch of DNA is variably repeated in each person
30
DETECTIVE DNA
father. However, a mutation during an early stage of embryonic development can create a band in the DNA profile of a child that has no match in the parental DNA. These mutations usually result in changing the number of the repeat segment in a VNTR by either increasing or decreasing the overall length of the VNTR. These probes are epecially crucial for paternity testing as analyzing several bands of the unique genetic signature gives a 'foolproof' evidence whether two persons are related or not. As the chances of matching of these several bands is higher in siblings and relatives and of course, highest in parent-child relationship, disputes related to parentage and immigration can be solved beyond doubt. Multi-locus probes are usually preferred in forensic identification where the tissue sample is available in plenty and good condition. Such probes, however, cannnot be used reliably in cases where the samples are mixed like in multiple rape, as several bands of the overlapping genetic profiles of different persons make it difficult to interpret the result. Thus, depending on which special enzyme is used to cut the human DNA and what kind of probe is used for the test, the DNA profile of an individual would have a specific pattern of bands. However, the genetic pattern in each case would be unique to that person. So, when the DNA profiles of two persons are compared it is understood that the two samples were treated exactly the same way, that is cut with the same enzyme and probed using the same detective. The first ever probe made by Jeffreys was a multi-locus probe as he had found two core sequences common to several repetitious segments at several loci in the total DNA. In India, this technology took root in 1988 by the pioneering efforts of Lalji SINGH and his team at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad. Interestingly, the probes developed by Lalji Singh are derived from DNA sequences first isolated from a poisonous female
31
Hinft M Kb A 231 2 C F 3 X 4
i frS6.5 1-34 3
l , J-1
2 . 3 *>.
a) DNA profile analysis by using a single-locus probe: On comparing the DNA profile of a child (lane 2) with that of the mother (lane 1), alleged father (lane 3) and an unrelated person (lane 4), it is revealed that the alleged father is not the biological father of the child. X denotes molecular weight marker DNA. b) DNA profile analysis by using a multi-locus probe : A child's DNA profile (lane 2) is compared with that of the mother (lane 3) and alleged father (lane 1). The alleged father, in this case, is the child's biological father as arrows indicate the DNA fragments which the child has inherited from him (Courtesy of Dr. Lalji Singh, CCMB, Hyderabad)
32
DETECTIVE DNA
Lalji Singh pioneered the DNA profiling technology in India. Banded krait (inset)
Indian s n a k e , Bungarus fasciatus, c o m m o n l y called the banded krait. Called Bkm sequences, they actually represent parts of the repetitive DNA present in the sex-determining region of the sex chromosome. Besides snakes, these have been shown to be present in many higher animals like birds, mouse and also man. The Bkm probe is a multi-locus probe that can detect several variable regions in the human DNA. It's conserved regions are long arrays of repeats of the fournucleotide sequence, namely, GATA. Many court cases mainly involving paternity disputes have been solved using the Bkm probe. Even single-locus probes have been developed by Lalji Singh and co-workers.
33
Scientists at the National Institute of Immunology (Nil), -New Delhi have also developed probes. One such comprises the cloned sequences of a highly variable region of the human minisatellite DNA. In yet another interesting development, it is seen that probes consisting of repeat segments occurring in the mitochondrial DNA help to conclusively prove a person's maternal relatives. This is so because mitochondrial DNA is inherited only from the mother. The probability of false association between unrelated individuals is extremely low using both multi-locus probes or well-constructed cocktail of single-locus probes. Using these probes, the infallibility of DNA profiling is such that two persons having identical DNA patterns is estimated to occur once in about 30 billion people a number that far exceeds the present total human population of five-billion! Identical twins are however, an exception.
he molecular Sherlock Holmes employed in DNA profiling technology is gifted with a special 'torch' before its detective ability is put to test. This torch always remains lighted up, that is at " o n " state. Well, a torch is needed at the first place for providing a visual proof of having found the few short matching sequences of DNA from a vast majority of unmatching stretches. This binding of the detective DNA to the complementary target DNA can be somehow likened to a swayambara in action, albeit at molecular level. However, unlike a princess who chooses her partner from among several prospective contenders by garlanding the lucky one, the detective on finding a complementary sequence of DNA stops and goes for a tight embrace. Some detectives (multilocus probes) however, bind to not one but as many complementary regions present in a DNA sample. It is to enable us to witness the final outcome of this molecular event that the detective is holding a lighted torch. The torch shines bright indicating the union. In technical parlance, the phenomenon of the fitting together of
Gifting the Torch
GIFTING THE TORCH 45
35
the probe DNA with the corresponding steps of the target DNA is called 'DNA hybridization'. This is brought about by the two complementary strands of DNA, comprising the double helix, holding each other tightly. Only temperature near that of boiling water is able to s e p a r a t e these strands. This process called 'denaturation' of DNA was at first thought to be irreversible until H A L L and SPIEGELMAN in 1960 discovered the phenomenon of hybridization. They found that so separated complementary single strands of DNA readily recombine, forming double helices if they are kept for an extended period at lower temperatures. This re-formation of the dou-
A DN A f
mentary single strand of DNA
Probe
t0,a
ble helix from base pairing of two single complementary strands, gave birth to the technique of DNA hybridization that has today revolutionalized molecular biology. Assume that the probe DNA has 10 nucleotides in the sequence: ATGGATGCTA. Then the complementary sequence it hunts for in the total DNA is TACCTACGAT. Binding of the two takes place even when one of the two complementary strands is immobilized on a solid support of a special filter paper. DNA profiling makes use of this property. The DNA segments from which the probe has to search out specific sequences that uniquely identify a person are immobilized and are single stranded. As the single stranded
36 Unwinding
DETECTIVE DNA
Heat Cool
Heat Cool
Heat Cool
Annealing
Changes in temperature can unwound and rewound DNA strands
probe gets hold of these target sequences, it forms a double stranded DNA. This union or hybridization is verified and made visible by the presence of lighted torch the detective is gifted with. The probe that has remained unbound is however, washed away before obtaining a visual record of the actual binding. But what exactly is this torch and how a small DNA strand is made to hold it? Well, the torch is actually a radioactive isotope which is simply a 'label' on the probe DNA. Radio-
GIFTING THE TORCH
45
37
P n ff a ace
Alpha
Beta Gamma
Radioisotope
Paper
Wood
Concrete
Alpha, beta and gamma radiations given out by radioisotopes have different penetrating powers. A Geiger counter (inset)
isotopes or radioactive isotopes are unstable elements which undergo random disintegration to produce different atoms. As a radioisotope disintegrates, it gives out energetic particles such as charged electrons or beta rays ((3-rays) or radiation such as gamma rays (y-rays). This radiation can be detected. Special devices such as Geiger Muller counter and scintillation counter can detect and quantify radioactivity in a given sample. However, to find exactly where a probe, labelled with a radioisotope, is located on a filter where it hybridized to its immobilized complementary strands, the filter is exposed to an X-ray film or photographic emulsion. The radiations that the radioisotope emits on decaying act on the grains of silver present in this film. This method is so sensitive that every single disintegration of the radioisotope giving out radiations can be detected as dark spots on developing the film. The result of hybridization of a probe with its
38
DETECTIVE DNA
target D N A is thus clearly visible as dark bands on the developed X-rays film. This is how a radioactive tag acts like a lighted torch whose presence and exact position can be visually ascertained. Many common biological elements are readily available in radioisotopically labelled form. For example phosphorus ( P), iodine ( I), sulphur ( S), and carbon ( 14 C) among others. Now for tagging on a radioisotope to a DNA strand or gifting it the torch a special technique is employed. This is known as 'nick translation'. However, a prerequisite for this reaction is the availability of the specific D N A strand that has to be labelled. This detective in the making is firstly produced by artificial synthesis or cloning once the sequence of the variable repeat segment useful in DNA profiling is found. For synthetic preparation, the target sequence needs to be fully defined. By automated chemical synthesis DNA strands up to 50 nucleotides long can be easily produced. The actual protocol of nick-translation reaction involves the mixing together of minute quantities of several chemicals. This is done on ice as some reactants are sensitive to high temperature. The DNA to be labelled is taken in a small plastic tube. Besides, the reaction mixture contains specific quantities of the radiolabeled (^Phosphorus-labelled) form of any one of the four nucleotides and the other three non-labelled nucleotides which are all commercially available in the nick-translation kit. The reaction of incorporating the 32 P-labelled nucleotide in the D N A strand, however starts only on addition of two enzymes: D N a s e I and D N A polymerase I. To start with, a 'nick' or break in a single strand of the double helix is introduced by DNase I. This enzyme produces random nicks in a DNA strand. Now acts DNA polymerase I. It actually catalyzes the addition of nucleotides, both unlab e l e d and radiolabeled ones present in the reaction mixture to only one side of the nick, that is actually the 3'-OH terminus. While doing so, DNA polymerase I removes from the
32 131 35
GIFTING THE TORCH
45 39
Nick
The first nucleotide on the 5' - phosphate side of the nick is removed.
A radioactively labelled nucleotide gets inserted in the DNA strand.
The process continues
A schematic representation of the nick translation reaction
other side of the nick (5' end), the existing unlabelled nucleotides of the DNA strand. This is by virtue of the inherent 5'>3' exonuclease activity of DNA polymerase I. Depending on the sequence of the DNA strand to be labelled, the labelled nucleotide finds an entry and gets incorporated in the double helix as the reaction proceeds. Thus, some of the existing unlabelled nucleotides in this double stranded DNA are replaced with their labelled counterparts. The sequence of nucleotides with radioactive tag added to the 3'-OH side of the nick is complementary to those in the opposite strand. The final product or the nick-translated DNA is an exact replica of the original DNA except for the several nucleotides tagged with the radioisotope. The exact amount of each reactant of nick-translation reaction may vary depending on the amount and length of DNA which has to be labelled. To understand the relative
40
DETECTIVE DNA
proportion of different reaction components used in nicktranslation, a probable set-up of this reaction is given below:
Nick translation reaction

Reactants Amount
DNA
0.5-1.0 |ig (ljig = 10"6 gram)
Mixture of unlabelled nucleotides (say, dATP, dGTP and dTTP)
5}il (1^1 = 10"6 litre)
oc32P-labelled CTP (50|xCurie/|xl)
5 (il
DNase I + DNA Polymerase I
5 |il
Distilled water to make total volume:
50 | . 01
The components of this reaction are gently mixed by tapping the tube and incubated at 15C for about an hour. This reaction is stopped by adding 5 (xl of stop buffer solution, that contains, ethylene diamine tetra acetate (EDTA).
GIFTING THE TORCH
45 41
The success of this reaction depends on the optimal conditions that include the correct balance of the amount of DNA, enzymes and free nucleotides together with conditions of temperature and time of reaction. It has been observed that smaller the amount of DNA added, greater is the percentage of its nucleotides being replaced by labelled nucleotides. Also, higher would be the total radioactivity or specific activity of the final product if instead of just one, more labelled nucleotides are added. By increasing the amount of DNA polymerase I, the rate of incorporation of nucleotides into the double helix increases. However, excess enzyme is not advisable since it may have unwanted activity of removing nucleotides. Similarly, excess of DNase I may cause unwanted nicks degrading the DNA probe. After stopping the nick translation reaction, the enzyme proteins present in the reaction mixture are precipitated by heating at 65C for 10 minutes. Finally, what is left is to separate the so formed probe having radiolabelled nucleotides from free nucleotides, both labelled and unlab e l e d and rest of the chemicals. This is done by using yet, another sophisticated technique known as 'gel chromatography'. Chromatography is a powerful and versatile technique used for separating and purifying the individual components of a variety of
mixtures The word 'chromatography' was Mikhail S. Tsvet, a Russian botanist who coined the word'chromatography'
42
DETECTIVE DNA
coined in 1906 by a Russian botanist, Mikhail S. TSVET to describe his separations of plant pigments. Generally, chromatographic separations involve three components: the packing material called the "solid phase", a solvent called the "liquid phase" and the sample to be separated. The cylindrical chromatography column, usually of dimension 0.7 cm x 20 cm is made of glass or plastic. It is packed with a permeable solid matrix immersed in a solvent. This matrix is actually made of beads of a cross-linked polysaccharide which may be dextran or agarose. These beads are inert but porous, thus allowing the passage of molecules through them. Commercially available beads called 'sephadex' are available in a wide range of pore sizes, (like
A mixture of large and small molecules applied to a column of dextran beads
Small molecules penetrate into the pores of beads Dextran bead
Lafge molecules emerge from the column first J Purifying the p r o b e D N A . Large probe D N A molecules separate out f r o m smaller free nucleotides by gel filtration c h r o m a t o g r a p h y
GIFTING THE TORCH
45 43
G-50 and G-100) making them suitable for separating molecules of various sizes and molecular weights. The sample containing the mixture of nick-translated DNA and other chemicals is placed gently at the top of the column tube. When a continuous flow of a solvent is pumped slowly through the column, the sample gets Washed down and different components of the sample exit the column along with the outgoing solvent at different times. Now, why do different components of the sample travel at different rates through the column? This is so because molecules which are small enough to enter the porous beads percolate inside successive sephadex beads as they travel. These beads, therefore, act like eddies in a river which slow the passage of smaller molecules. The larger molecules remain between the beads and hence move faster through the column, emerging out first from the bottom of the column. As the nick-translated DNA strands are bigger molecules than unincorporated nucleotides, they come out of the column before the latter do. Thus, the probe DNA is purified on separation from free nucleotides. About 20 fractions of 8-9 drops each are collected in clean tubes. The radioactivity in each of these fractions is monitored using a hand-held device, the Geiger counter. Further, a small amount (5 |il) is removed from each fraction and added to 2 ml of scintillation fluid put in special bottles. The radioactivity in each fraction is quantified using a scintillation counter. The fractions with highest radioactivity are pooled which is now taken to be the probe DNA. The specific activity of the probe should be normally around 108 cpm/jxg of DNA. This means that the torch light should be strong enough to be recorded for visual analysis. It however, depends on the extent of nucleotide replacement during the reaction. The detective is thus ready to render its services. Till then, it is stored at - 20C. However, before this probe is put to its job,
44
DETECTIVE DNA
Pooling the fractions from a chromatography column containing the purified probe DNA
it is heated at 100C for 5 minutes to separate the two strands of the double helix. Although some other methods of radiolabelling are also known, nick-translation is the most commonly employed technique for tagging a radioisotope to a DNA strand. But why a radioactive isotope is chosen as a label for making a DNA probe? Generally, a probe is radiolabeled since the specificity of such probes is as high as about 98%. The sensitivity of radiolabeled probes is again very high, almost 100%, as they can accurately detect even a tiny target sequence. However, the drawbacks are the associated operational difficulties and health hazard as any direct contact of the body
GIFTING THE TORCH
45 45
with radioactive substance may prove harmful. Fresh probes are preferred since they have short shelf life. Also, the entire protocol detecting specific DNA segments in human DNA using radiolabeled probes is time-consuming taking about 2-3 weeks. The high cost of the sophisticated equipment required for such a laboratory is yet another unfavourable factor. In this light, non radioactive probes based on enzymatic detection of the specific DNA sequences are being developed. The most promising of such techniques is one that uses biotinylated probes. Here the probe is tagged with a molecule of 'biotin' whose presence can be determined by enzyme-labelled molecules of 'avidin' which very specifically bind to biotin. On adding an appropriate substrate for the enzyme, a coloured product is formed which indicates hybridization of the DNA strands. Besides biotin, even a steroid molecule named 'digoxigenin' may be used as a tag. Similarly, some chemiluminiscent substances can be used to label a DNA strand. The main advantages of such probes are that the results are obtained faster besides these probes are safer and cheaper substitutes to radiolabeled ones. They also have a longer shelf life and need not be prepared fresh each time. Moreover, standard laboratory facilities are good enough for carrying out/tests using non-radiolabelled probes. The sensitivity of these probes is, however, less than the radioactive counterparts. Nevertheless, in forensic identification, it is the radiolabeled detective that finds most common usage.
The Master Plan
f I lommie Lee Andrews was at last brought to book in 1987 J L for committing sexual assault of two women in Orlando, USA. This crime is known till today as Andrews was the first man to be convicted in USA on basis of DNA profiling technology. The first assault occurred in May 1986 on Nancy Hodge. The second victim, also a young woman, was assaulted a few months later. The culprit, although cautious enough not to leave any evidence of his presence at the place of crime, could not, however, erase the cells of his body that were later found in the vaginal swabs of the two victims. Carrying his molecular identity card, these tiny cells proved conclusively that Andrews was the culprit. However, this DNA evidence was sought to nail the culprit only when standard forensic tests could not yield any foolproof evidence for the same. Moreover, the potential of DNA, profiling dawned upon Andrew's prosecutor as detective DNA had started going places after its heroic triumph in catching the culprit- of a similar case in Leicestershire, UK. Now, what exactly must have been done for carrying out this
THE MASTER PLAN
47
DNA analysis? Well, for a thorough understanding of DNA profiling, the entire step-wise protocol or the master plan behind this technology should be clear. This master plan involves several biotechniques, recipes for which have been standardized. These are carried out one after the other in a proper sequence, the end result of which yields a DNA profile. The role of the detective DNA is very crucial as it is the key player in this master plan. To start with the prerequisite for producing a DNA profile is the availability of any biological specimen. The tissue sample could be any body cell, as every single cell of a person's body contains exactly the same genetic information. However, most commonly available samples are blood, hairs with root cells, and semen. In the two assault cases of Orlando, the culprit's tissue sample was semen from which the male reproductive cells called spermatozoa or sperms were recovered. From these cells DNA was extracted for profiling. Basically, DNA extraction from any cell involves the disruption of cells by the breakdown of the outer cell membrane. Nuclei, the cellular compartments that house the genetic material, are then broken down to release the blueprint. However, before this the unwanted cellular debris is separated from the nuclei by spinning the solution at high speed in a machine called centrifuge. A special detergent called sodium dodecyl sulphate (SDS) is then used for releasing the DNA contained in the nuclei. This detergent dissociates the proteins from the DNA. Subsequently the proteins present in the solution are degraded using an enzyme called proteinase k. Also, treating it with the enzyme RNase specifically degrades the ribonucleic acid (RNA) present in the solution. The so obtained DNA is then purified from the degraded proteins, RNA and rest of the cellular components by repeated washings with phenol and chloroform. In this, the mixture of DNA and these chemicals is shaken apd the two separate out as distinct phases by spinning in a centrifuge. As proteins are soluble in organic phenol and chloroform, they are separated
48
DETECTIVE DNA
Centrifuge
-RotorI
Vacuum Refrigeration Motor
(a) Centrifugal force -
(b>
(c)
(d)
(e)
Solvent Small sized particles Medium sized particles
Time of centrifugation
Large sized particles
Depending upon their size and density different sized particles separate out as distinct layers when rotated at a very high speed in a centrifuge
THE MASTER PLAN
49
from DNA which is soluble in the aqueous phase. The DNA present in this aqueous phase is precipitated with ethanol (alcohol). This technique may be suitably modified for extracting DNA from different body tissues. The DNA extracted from a cell for analysis actually consists of very long fragments. These are, therefore, chopped into shorter and discrete fragments which are more manageable. For doing so, special enzymes called restriction endonucleases are employed. These wonderful molecular scissors occur naturally in several bacteria where they safeguard the DNA of the host by degrading and hence making ineffective any invading foreign DNA molecules. This significant piece of technology came as a major breakthrough in molecular biology way back in 1978 when two A m e r i c a n s c i e n t i s t s , H a m i l t o n S M I T H and D a n i e l NATHANS discovered the restriction enzymes. Interestingly, these important molecular tools recognize and cleave only a specific sequence of four or six base pairs long. It is like a colour blind person who can see only specific colours. The sequences recognized are often "palindromic", that is, the nucleotide sequences of the two strands are the same in the recognized region. Both the strands of the double helix are thus cut within the recognition site. However, the same sequences if present in the DNA of the host remain unseen by the enzyme as they are "camouflaged" by the presence of a methyl group (-CH3) in the base adenine or cytosine of that DNA sequence. Over 200 restriction enzymes have been purified from different species by bacteria. For example the enzyme Eco RI is obtained from Escherichia coli and Hind III from Hemophilus influenzae. Each enzyme recognizes a different specific sequence. Like Eco RI recognizes only GAATTC and chops down D N A at just that sequence. The smaller fragments produced are called 'restriction fragments.'
50
DETECTIVE DNA
Recognition and cleavage site
Bam HI
Hae III
Hamilton Smith (left) and Daniel Nathans discovered the restriction enzymes the wonderful molecular scissors which recognize a specific sequence of bases and cleave the DNA there
Given the unique genetic signature of every person, a particular restriction enzyme produces fragments of different lengths in each one of us. This is because of the presence of short non-coding sequences spread over the genetic material which are repeated a variable number of times in different individuals. The length of these variable number of tandem repeats or the VNTR's thus differs from person to person,
THE MASTER PLAN
51
which can be determined if a restriction enzyme makes a cut ai or near the sites flanking that segment. In other words, cleavage on either side of the V N T R generates restriction fragments that differ in length which in turn reflects the number of repeating units it has. However, the size and number of these fragments would vary if a different restriction enzyme is used or more than one enzyme is made to cut the DNA. These restriction sites should actually provide a uniformly inherited genetic marker for that particular V N T R segment. This enzymic treatment usually carried out at 37C reduces the long DNA fragments
Molecular scissors
DNA fragments
Cleavage
Noodles
A long strand of DNA cut into several randomly sized pieces of DNA could be likened to the varying lengths of noodles present in a soup
52
DETECTIVE DNA
into several randomly sized pieces of DNA. It is something similar to a soup having varying lengths of noodles in it. For assembling the D N A f r a g m e n t s l e n g t h - w i s e , the technique of gel electrophoresis is e m p l o y e d . The n a m e ' e l e c t r o p h o r e s i s ' is d e r i v e d from two Greek words namely, 'elektron' and 'phoresis' (carrying). It describes the principle that different molecules migrate at different rates
Arne Tiselius won the Nobel Prize for his breakthrough on developu n d e r
i n f l u e n c e o f
electric
ing electrophoresis the premier charge. The potential of this method for separating and technique was brought to light
analyzing proteins and nucleic acids j n 1 9 3 7 b y t h e p i o n e e r i n g ef-
forts of Arne TISELIUS, a Professor at Uppsala University, Sweden. Using electrophoresis he separated the human serum into four classes of proteins, a b r e a k t h r o u g h for which he won the Nobel Prize for chemistry in 1948. This technique is an essential tool used extensively in molecular b i o l o g y for separating and analyzing nucleic acids and proteins. The gel used in this technique is made of agarose, a jelly-like material derived from Agar agar found in a
Structure of agarose gel
THE MASTER PLAN
53
Electrnda DNA fragmen ts Agarose gel Electron
Buffer solution
Electrophoresis
chamber
Small DNA fragments move faster through gel than large ones
DNA fragments are size sorted by agarose gel electrophoresis. Loading an agarose gel (inset)
sea weed called kelp. Usually 0.8% agarose is used. It is heated and spread on a long horizontal plastic plate, thus molding it into a thin slab. Small wells are formed at one end of the gel by placing a plastic comb in the hot gel before it cools and solidifies. The soup of DNA fragments is then gently placed
54
DETECTIVE DNA
Ethidium bromide molecule
DNA fragments on the gel appear to glow as distinct size-sorted bands when stained with a fluorescent dye, ethidium bromide and viewed under UV light. Intercalation of ethidium bromide with DNA (inset)
in these wells using special micropipettes. When an electric charge is applied across the gel, the D N A fragments migrate from the negative (cathode) to the positive terminal (anode) at speeds dependent on their respective sizes. Smaller D N A fragments thread their way faster through the complex network of gel molecules while large D N A fragments linger behind. Samples are usually run at 3-4 Volts/cm for several
THE MASTER PLAN
55
hours. The gel actually acts like a molecular sieve. The end result is, understandably, a neat separation of DNA fragments based on size. What is actually obtained is simply a D N A smear! At this stage, the smear can be visualized by staining the gel with a DNA-specific stain, which is a chemical called ethidium bromide. D N A on the gel can be seen as glowing bright bands under the ultraviolet light. Nevertheless, for detecting specific target sequences or the variable repeat segments huddled somewhere within the D N A smear, another essential technique that has revolutionized D N A analysis is used. This technique first perfected in 1975 by Edward M. SOUTHERN, a biologist at the University of Edinburgh, is called Southern blotting. The technique essentially involves the transfer of DNA smear from the fragile, wobbly gel to a more firm support. The process is quite similar to soaking up of ink by a blotting paper. A variety of special blotting membranes are available, such as nitrocellulose, charged or uncharged nylon and polyvinyl difluoride (PVDF) membranes. Nitrocellulose filter is, however, most commonly used. Before blotting, the helical zip of the double stranded fragments in the gel is opened out, separating the two single strands. This is done by treating the gel with a mild alkaline solution. In Southern blotting the gel carrying the DNA smear is first placed flat on a " w i c k " of a special filter paper called Whatman 3 M M which is connected to a trough containing a concentrated solution of the chemical, saline sodium citrate (6 x SSC). No air bubbles should be trapped underneath the gel. A sheet of nitrocellulose filter, cut to proper size, is then carefully placed on top of the gel without trapping any air bubbles. The top edge of the filter should be in line with the top of the gel. Subsequently, a large stack of dry absorbent paper towels are laid flat on the filter. This set-up is left undisturbed for about 36 hours while changing the paper towels from time to time.
56
DETECTIVE DNA Southern blotting
DNA is trapped
Nitrocellulose
filter
Gel
Fluid flow
Filter paper soaked in salt
A nitrocellulose filter traps the DNA molecules just as a blotting paper blots ink
The absorbent paper towels draw up the salt solution by capillary action which of course, passes via the gel. On passage of the liquid through the gel, the single strands of D N A in the gel are swept out only to be trapped by the parched nitrocellulose filter sitting on top of the gel. A perfect replica of the DNA smear is thus obtained on the filter, faithfully preserving the unique pattern of the cut DNA fragments. There is absolutely no smudging of the size-sorted fragments during this transfer. The DNA fragments are then bound permanently to the nitrocellulose filter by baking it in an oven. This smear is not visible on the filter. A detective is now employed to 'probe' these DNA fragments for target sequences.
THE MASTER PLAN
57
Weight Glass plate Paper towels Blotting paper Nitrocellulose filter Gel Support Cellophane 6x Saline sodium citrate 3mm paper Tray solution
The Southern blot apparatus for the transfer of DNA from agarose gel to a nitrocellulose filter. A gel set-up for blotting (inset)
The detective is a specially prepared one as its base sequence is complementary to the target variable repeat segment. Therefore, it selectively binds to that particular V N T R by the process of hybridization. Usually a multi-locus probe or a combination of two or more single-locus probes is employed for making a DNA fingerprint. The probe D N A is made single stranded by heating the double helix at 100C for five minutes. Before actual hybridization, the nitrocellulose filter is kept in a prehybridization solution at 42C for about 16-26 hours. This solution contains several chemicals which actually bind to the nitrocellulose filter blocking the background from any non-specific binding of the probe DNA.
68 DETECTIVE DNA Collection of body specimens
Southern blotting Radioactive probe I DNA at work ,
Extraction of DNA
!Bound probe visualized by exposure to X ray film. Isolated DNA cut into fragments DNA fragments size-sorted
DNA fingerprint
Steps to a genetic signature
THE MASTER PLAN
59
The most crucial part of D N A profiling comes now. The filter carrying the exact replica of the DNA smear firmly adhering to it is bathed in a specially prepared hybridization solution containing the single stranded probe DNA. For this, the filter is usually placed in a plastic bag which can be sealed from top using a heat-sealing device. Hybridization m a y be carried out at temperatures varying from 42C to 65C for about 16 hours, depending on the stringency of conditions required. Carrying the torch of a radioactive isotope, the detective starts on its hunting spree searching untiringly for complementary sequences from those lying silent on the filter paper. On finding them, the detective promptly binds to those sequences forming a double helix and, consequently, gets stuck on to the filter. As the length of a particular variable repeat segment varies from person to person, the detective, therefore, binds to a segment of specific length present in the D N A smear of an individual. After hybridization, the excess unbound probe is washed away with a series of solutions at usually 65C for 30 minutes. The solution contains the detergent, sodium dodecyl sulphate (SDS) and the chemical, saline sodium citrate (SSC). Thus, only those fragments to which the detective had got bound remain labelled with the radioactive torch. To obtain a visual record, the nitrocellulose filter is subsequently pressed against an X-ray film placed in special X-ray cassettes. The radioactive isotope tagged to the probe is the detective's torch which exposes only those parts of the lightsensitive X-ray film which come in contact with hybridized fragments on the filter. This is done in a dark room. The cassette is then placed undisturbed in a -70C freezer. The exposure period may range from a few hours to a couple of days. On developing the X-ray film an autoradiograph or autorad is obtained. Having a bar code appearance, an autorad has a series of thick and thin dark sooty bands at specific locations which indicate the target, variable repeat sequences searched by the
60
DETECTIVE DNA
DNA strands 1
2
3 4 5 6 DNA strands are invisible
DNA fingerprint
The probe searches the complementary DNA strand and binds to it
The detective at work. Cassette for exposing the nitrocellulose filter to an X-ray film (inset).
THE MASTER PLAN
61
detective. Rest of the X-ray film has an off-white shade. This is how the original uninterpretable smear of D N A fragments is converted into a well defined DNA profile of a person. Every individual has a unique bar code pattern of his/her D N A profile. Therefore, in technical parlance, it is called forensic restriction fragment length polymorphism (RFLP) testing. For matching the DNA profiles of two biological samples, the positions of all the bands of one is compared with the band pattern of the other. The size of a DNA fragment in a particular band is determined by comparing the distance moved by that band relative to bands of D N A fragments whose lengths are already known. The latter is called the 'control' or 'marker' D N A which is run on the same gel along with the D N A sample to be profiled. The entire master plan for making a DNA profile, right from extracting D N A to obtaining the autorad, takes about two to three weeks. Successfully hybridized filters can be rehybridized with a different probe to generate a new D N A profile. For this, the earlier probe bound to DNA fragments on the filter is washed away using certain chemicals. This may be repeated for probing with yet another detective. Andrew's crime became evident in both assault cases on comparing the DNA profiles of his blood, the victim's blood and the smear obtained from the vaginal swab of the victim. The D N A profiling tests in this case were performed by a team of scientists led by Michael BAIRD at the Lifecodes Laboratory in Valhalla, New York. As the vaginal swab contained cells of both the victim and the culprit, it produced a mixed molecular signature of two persons. However,- on comparing it with the D N A profile of the victim, the mismatches observed were understandably the genetic fragments of the culprit. The relative position of these mismatching bands actually perfectly matchedband for bandwith that obtained from Andrew's blood. These
62
DETECTIVE DNA
Suspects
Criminal
Nailing the culprit: Matching the DNA profile of a criminal with those of suspects
amazing results created a no-escape situation for the criminal. Besides, in forensic identification several cases relating to parentage dispute have been logically concluded in the West and also at home. The detective, in each case has cleverly brought to fore the actual biological mother and father of a child as a child shares half of the bands of his/her D N A
THE MASTER PLAN
63
A DNA pattern (centre) of blood stain obtained from the site of crime compared with those of seven suspects. The one on its immediate left is the exact match
profile with each parent. DNA profiling gives a conclusive proof of parent-child relationship as the samples would simply match or they would not. In an interesting immigration case, a Ghanian boy left his mother in UK and emigrated to Ghana where his father was staying. However, his decision later to return back to his mother proved difficult as immigration authorities suspected him to be the son of his mother's sister who also stayed in Ghana. Conventional tests showed that the boy and the lady
64
DETECTIVE DNA
were closely related but they did not rule out the possibility that he was her nephew. In stepped the molecular detective to help in revealing the true identity of the boy. Genetic analysis of blood samples of the boy, the putative mother and her other three children by the same man produced their individual molecular fingerprints which were compared for
Proving a family relationship using a multi-locus probe. Several matching bands in DNA profiles helped Andrews, a Ghanian boy immigrate to England to join his mother and siblings. Lane X shows an unrelated person
THE MASTER PLAN
65
relatedness. However, neither the boy's father nor any of the mother's sisters were available for the test. The result showed that many of the fragments of the boy's DNA profile matched with those of the other three children proving that all were of the same father. Also, several other bands of the boy's genetic signature found a perfect match with those of putative mother's profile. As the occurrence of such a match of D N A profiles of two individuals is possible in no other relationship but parent-child, it was proved beyond doubt that the boy was indeed the woman's son. Thus, D N A profiling is a powerful technology comprising an array of most sophisticated modern biotechniques. In forensic identification, it conclusively establishes the identity of a person by employing a molecular detective.The precision of the technology is such that a person whose any biological specimen is available has actually handed over, without an inkling, his identity card with all his credentials name, address, telephone number.... and blood group a sufficient evidence to accurately identify him.
From One To Many
otwithstanding the vast potential of DNA profiling, this powerful and promising technology is incapacitated if the DNA recovered from a biological specimen is too little or highly degraded. Fortunately, in the year 1985, a revolutionary technique called Polymerase Chain Reaction (PCR) came to its rescue. Invented by US scientists led by Kary MULLIS in Henry ERLICH'S laboratory at the Cetus Corporation in Emeryville, California, this technique helps to amplify small pieces of DNA very selectively. The technique plays a significant role in giving a practical dimension to DNA profiling. Using this ingenious technique DNA fragments, as small as a single molecule of DNA, can be amplified many times and analyzed. PCR is, therefore, ideally suited where the quantity of biological specimen available is very low such as a single hair strand or a tiny blood stain left at the site of a crime.
PCR actually mimics the natural way of synthesis or replication of DNA. It is a test tube method of amplifying DNA where the two strands of DNA are first separated out by heating at 95C for 15 seconds. This process of heat denatu-
FROM ONE TO MANY
67
ration, unwinds the double helix to be copied into two single strands. These strands now act as what is called the 'templates' for the synthesis of two new complementary strands, which specifically bind to them. For this process, an enzyme called D N A polymerase is employed which runs along the template strands building up the new chain using the free nucleotides available to it. The double helical structure of D N A is thus restored in both the single stranded templates.
Parent DNA
Template
New DNA chains
Daughter DNA molecules
A replicating DNA molecule
68
DETECTIVE DNA
But, how does the enzyme know from where to start making a new strand? For directing the enzyme to begin its action, short pieces of DNA flanking or present on either side of the region to be amplified are added. Known as 'primers', these sequences of DNA having about 20-30 bases, are of primary importance and a major requirement for carrying out PCR. These are usually artificially synthesized as their nucleotide sequence is known. The length of the amplified D N A product is equal to the length of the two primers plus the distance between the primers. Thus, the various ingredients of PCR are the polymerase enzyme, the four building blocks of DNA the nucleotides, namely ATP, GTP, CTP and TTP, the primer DNA strands and the D N A to be amplified. After separating the two strands of the double helix the reaction mixture is cooled to 37C. This allows the primer strands to bind to the complementary sequences on the template DNA which has to be copied. The enzyme acts now. It works in only one direction from a dpuble stranded starting point which is the end of each template. It then moves along each template strand assembling the appropriate nucleotides, thus forming a brand new complementary strand. With this, one cycle of PCR is completed. So, starting from two complementary single strands of DNA, at the end of the first cycle of PCR, two double helices are formed which are actually identical to the original piece of DNA. For the second cycle of D N A synthesis to begin, the temperature of the mixture is again raised for separating the two complementary strands of the double helices formed in the previous cycle. The resulting single strands now act as templates to which the primers bind as soon as the reaction mixture is cooled once again. Thus, a second round of PCR cycle begins. The technique therefore, involves the repeated cycles of DNA synthesis or replication where the product of the first cycle becomes the template for the next cycle. Understandably, with each cycle, which is of about five minutes, the amount of DNA in the sample is doubled. In just 25 such
FROM ONE TO MANY Target DNA
69
Reaction mixture DNA
m , polymerase
Primer Reaction mixture heated to unwind target DNA.
First cycle
Primers initiate the synthesis of two new DNA chains Two copies of target DNA J
'DNA unwinds Second cycle
Primers bind
New DNA chains formed
Four copies of target DNA A m p l i f y i n g D N A using the p o l y m e r a s e chain reaction
70
DETECTIVE DNA
rounds of PCR, more than a million copies of a D N A segment are produced. This million-fold amplification of a target D N A sequence takes only about 2-3 hours. The original PCR developed in 1985 was slow and expensive as heating in this technique inactivated the enzyme D N A polymerase I obtained from Escherichia coli. So, fresh enzyme had i o be added for each cycle. In 1987, Cetus scientists isolated a thermostable DNA polymerase from a bacterium, Thermus aquaticus. This heat loving (thermophilic) bacteria naturally thrives in hot springs and geysers. The enzyme has been appropriately named Taq polymerase. Thus, the D N A polymerase from E. coli was replaced with Taq polymerase in 1988. Fresh enzyme is, therefore, not added during each cycle as this enzyme remains functional even at the heat denaturation step of PCR. This has enhanced the commercial appeal of the technique. An automated PCR machine where temperature of the sample mixture can be controlled has been developed. It is known as the DNA thermal cycler, which is indeed the DNA photocopier! After amplifying the DNA with PCR, the product is analyzed by gel electrophoresis. The amplified DNA is actually a thick single band on the gel which can be stained with ethidium bromide and seen under UV light. Thus, using PCR a desired DNA sequence can be obtained in pure form and a b u n d a n t quantity. It is basically a t e c h n i q u e by w h i c h DNA can be amplified without cloning which is a tedious, timeThermal cycler The PCR machine consuming method.
FROM ONE TO MANY
71
The bacterium, Thermus aquaticus inhabits the hot waters of such geysers
Nevertheless, PCR is not free from hurdles. Firstly, the very sensitivity that makes PCR so useful, actually threatens to render it useless if a contaminant DNA is present along with the sample to be amplified. The reason is that although the primers bind to specific sequences they cannot distinguish sample D N A from unwanted contaminant D N A which might, by chance, be having a sequence complementary to that of the primer used in the reaction. Therefore, utmost care has to taken to ensure that no stray DNA molecules enter the reaction mixture as they could also be amplified simultaneously. Secondly, primers used in PCR must be long enough to avoid the possibility of their sequence to occur in a contaminant DNA. Also, the amplification capacity of the reaction is not unlimited. This is due to the accumulation of the double stranded product. The enzyme also becomes insuffi-
72
DETECTIVE DNA
cient at later cycles of PCR as there occurs an excess of templates. Another pitfall of PCR is the misincorporation of nucleotides which occurs mostly if the DNA to be amplified is degraded. Although the technique is, at present, expensive to carry out, the cost might drop due to its popularity and host of applications. PCR is a tremendously versatile technique for it finds immense use in several fields. The spectrum of applications of PCR include areas as diverse as forensic identification, medical diagnostics, food analysis and molecular biology research. Once the PCR amplified DNA is in hand, it can be analyzed basically in three ways. First, a DNA profile can be made. For forensic identification, however, if the base sequences flanking a variable repeat segment or a VNTR are known then primers complementary to these regions can be synthesized for PCR. These primers would selectively amplify only the particular repeat segments from tens of thousands of other such regions in the genetic material. The length of this repetitive D N A segment is then determined by gel electrophoresis which would be different for different individuals. For obtaining a visual record it can be either stained with ethidium bromide and the bands seen under UV light or the size-sorted PCR products are transferred from agarose gel to a nitrocellulose filter by Southern blotting. Further, it is probed with a detective having a sequence complementary to that of the repeat sequence. A person who is heterozygous for that D N A sequence will show two distinct bands, one inherited from the father and other from the mother. Whereas a homozygous individual will show a single band for that particular variable repeat segment. The second way of analyzing the PCR product is by simply blotting the amplified DNA directly on a filter. The blotted D N A looks like a dot on the filter. The target repeat segment in this blotted DNA is identified by hybridizing it with a
FROM ONE TO MANY
73
PCR amplified DNA blotted on nitrocellulose filter
Biotinylated PCR amplified DNA*
probe
Substrate
Biotin Streptavtdin Biotin ]
Enzyme
Coloured product
Biotin
Nitrocellulose
filter
A coloured dot appears if the probe binds to a matching sequence
PCR based dot blot assay using biotinylated probe DNA
74
DETECTIVE DNA
probe having a sequence complementary to the repeat segment. This test is called the 'dot blot' hybridization. The probe used in such PCR based assays are usually labelled with non-isotopic substances. Thus, forensic identification based on PCR bypasses the laborious and time-consuming techniques involved in D N A profiling or RFLP testing. Thus, besides being highly specific and sensitive, PCR based assays are very rapid. However, to detect more than one variable repeat segment each present at a specific site or locus on the genetic material, the PCR based dot blot assay has been appropriately modified to the reverse dot-blot format. In this, several single-locus probes, each detecting a different variable DNA segment, are first blotted and fixed separately on a nitrocellulose filter. Interestingly, these probes are not labelled. In fact, this approach uses biotin-labelled primers in the PCR. Thus, the amplified D N A which has to be tested for forensic identification is labelled with biotin. For detecting hybridization between the amplified DNA and immobilized probes, a substance called streptavidin labelled with an enzyme, namely, horseradish peroxidase is added. Streptavidin binds highly specifically to biotin. Subsequently, on adding an appropriate substrate, a 'blue' coloured product is formed. Thus, a blue dot appears on the filter which indicates hybridization. The first commercially available DNA profiling kit employs the reverse dot blot hybridization technique which is an assay based on PCR. This kit introduced by Cetus scientists in February 1990 reveals in each individual the unique pattern of inheritance of a variable repeat segment at a specific locus in our genetic material called the H L A - D Q - a locus. A yet third way of analyzing the PCR amplified D N A is by finding the sequence of nucleotides of that segment using special D N A sequencing techniques. This explains why PCR is being extensively used to locate in an individual changes in the nucleotide sequence of genes coding for vital proteins.
FROM ONE TO MANY
75
Cause of several genetic defects has thereby come to light. Diagnostic tests for several genetic disorders using special detectives and requiring specific PCR amplified D N A segments from an affected person-are in the offing. Infectious diseases can also be diagnosed by amplifying specific segments of the disease-causing organism which could help identify it. With the advent of PCR, it has now become possible to provide clues to our genetic history. Dating bones by profiling PCR amplified DNA recovered from bones is now a reality In an interesting application of PCR, a tiny amount of D N A was extracted from the bone remains of body of a murder victim found w r a p p e d in A carpet. This D N A extracted by Erika HAGELBERG in 1989 at John Radcliffe Hospital, Oxford was sent to Jeffreys for profiling. Facial reconstruction had shown that the body was of a young boy named Karen Price who had disapp e a r e d from a children's home eight years ago. A l t h o u g h the sample was Dating bones by profiling PCR amplified highly contamibone DNA
76
DETECTIVE DNA
nated with microbial DNA, Jeffreys could amplify six variable repeat segments using PCR. On comparing these D N A fragments with those from Karen Price's parents, it was conclusively proved that the body was of Karen Price. Thus, for the first time in 1991, a British Court accepted the PCR technique for providing forensic evidence. In other words, the technique got official approval as a forensic tool, t h e detective used for this bone DNA profiling was a DNA sequence complementary to a highly variable repeat segment. PCR has indeed paved the way for the study of a new field namely, molecular archaeology. Ancient DNA from fossil remains of Egyptian mummies dated to be 2400 years old has been extracted and amplified using PCR by Svante PAABO, a biochemist at the University of California, Berkeley. Degraded DNA from tissues of extinct animals as old as 13,000 years like the giant ground sloth besides those from the fossil remains of the wooly mammoth, a close relative of the modern elephant, and the quagga, a zebra-like animal have been amplified by PCR. Thus, PCR is a remarkable tool that is helping to chart the evolution of many species. For tracing the evolution of human populations, scientists have been studying variations in the mitochondrial DNA using PCR. Mitochondrial DNA is inherited only from the mother. Such efforts have concluded some interesting findings, that is, all human beings are descendants of the same woman who lived in Africa about 200 thousand years ago. Popularly, our common ancestral mother is called the "mitochondrial Eve". In an interesting finding, Adrian Targett, a school teacher is suggested to be a direct descendant of the 'Cheddar Man', a 9000-year old skeleton discovered in Cheddar Gorge, UK. The DNA sample from Targett was found to closely match the mitochondrial DNA taken from Cheddar Man's molar teeth, suggesting that the two had a common maternal ances-
FROM ONE TO MANY
77
(c)
(a)
(b)
Glimpse of ancient DNA : Degraded DNA from Egyptian mummies (a) and fossilized extinct animals; wooly mammoth (b) and quagga (c) have been amplified by PCR
78
DETECTIVE DNA
m^^m^^mm^mMm^SBSBmat _ _
The skull of Cheddar man
tor. Targett's ancestor is believed to be a hunter-gatherer. For this study, scientists compared the DNA of Cheddar Man with D N A samples of people whose families had lived in the Cheddar area for many generations. In yet another breathtaking discovery in 1990, a team of American scientists led by Edward GOLENBERG, at the University of California have extracted, as they claim, the oldest D N A in the world which was amplified using PCR. This D N A was extracted from a fossil leaf of an extinct plant species, Magnolia latahensis. Excavated from sediments of a lake in Clarkia, the fossil has been estimated to be 17-20 million years old. It is however, intriguing that how D N A from such fossils has still survived? The environmental conditions seem to account for this neat preservation. Ancient fossilized organic material trapped in ambera yellowish resinous product of some trees, has also been seen to~betb,eutifully preserved. Insect fossils protected in amber ftave been- ; obtained. Steven Spielberg's mega hit movie,
FROM ONE TO MANY 89
Fossil Magnolia leaf
Jurassic Park based on Michael Crichton's best seller, is a take off on the exciting extension of this technology. The movie is about recreating dinosaurs from the DNA of these extinct animals extracted from blood sucking insects trapped in amber. Seeing the immense potential of the powerful iechnique of PCR, it seems that several untouched pastures are vet to be explored where a p p l i c a t i o n of this technique could pay rich dividends.
,. . r i i
Genetic secrets captured in
amber
Detecting Defective Genes
es. We are all different. Each one of us has a unique set of distinctive characteristics, however minute be the differences in our genetic material. A majority of these differences in our blueprints are those which actually do not have any significance in affecting our general well being. These are the short tandemly repeated sequences sprinkled all over the blueprint. Present as junk D N A they do not code for any vital protein. Such sequences have been judiciously exploited in individual identification as the number of times such a sequence is repeated varies from person to person.
Other differences in the blueprint are understandably those which influence in innumerable ways the physical and mental make-up of a person. These are the e s s e n t i a l regions of D N A which code for vital proteins. This explains why you have straight hair and black eyes while your friend has curly hair and blue eyes. There are, however, other types of differences which, albeit few, are known to have disastrous effects on the carriers of such differences condemning them to a life
DETECTING DEFECTIVE GENES
81
of disease and suffering. In other words, such changes in the genetic material affect the functioning of a gene coding for a vital protein which is physically manifested by the appearance of a genetic disorder. At present about 4000 human genetic disorders are known and understood at molecular level. The ill-fated alterations that occur in the genetic material causing these disorders are called mutations. These are sudden inheritable changes in the
mRNA Codons
Peptide chain
-Amino actds Peptide bond
Protein
Proteins are vital molecules which contain the decoded genetic information
82
DETECTIVE DNA
blueprint which usually occur during replicaton of D N A or may be spontaneous by nature. Although D N A faithfully copies itself, this marvellous molecular feat may falter unpredictably leading to permanent changes in the genetic instructions coded in these molecules. Thankfully, such errors are extremely rare as they might not occur at even the millionth time "of replication in a row. Their occurrence is, therefore, merely a matter of chance. This accidental mistake could occur at a single point, that is, due to a single base change which is known as point mutation, or a stretch of D N A may be altered. The latter may be simply due to addition or deletion of some bases or there could be an inversion or translocation of specific stretches of DNA segments of genes coding for vital proteins. The proteins coded by these imperfect, defective genes are hence different and mostly faulty. These altered proteins manifest in the body causing fatal physiological changes associated with that disorder. Now, how exactly are such mutations in a gene detected for specific diagnosis of a particular genetic disorder? The basis of detecting these defective genes lies in the fact that gene alterations, in many cases, fortunately leads to creation or elimination of recognition sites for one or more restriction enzymes, the molecular scissors that cleave D N A at specific regions. The mutated gene or the DNA segments flanking that mutated gene may, therefore, contain a site for a restriction enzyme which is absent in the normal gene. It could also be possible that a restriction enzyme site within a normal gene or present close to ('linked' to) a normal gene may be eliminated by certain mutations. This only means that different sized D N A fragments would be produced if that particular restriction enzyme is made to cut the DNA of a normal person and that of the affected individual. Sorting these fragments by gel electrophoresis followed by Southern blotting and hybridization with a specially designed detective would then provide a visual record a bar code pattern or
83
Gel
electrophoresis
A B C D
A X
A change in DNA that affects the site of a restriction enzyme is detected by difference in lengths of the cut fragments
the RFLP pattern which would be different for normal and affected individuals. It thus reveals the different sized fragments in a normal gene and its mutated counterpart. This wonderful diagnostic tool based on DNA profiling technology uses specific radiolabeled D N A segments of the affected gene as detectives. If a cleavage site of a restriction enzyme is lost or created within the gene itself, diagnosis on the basis of different RFLP patterns could be confirmed. However, if a region flanking that gene has an altered cleavage site of a restriction enzyme,
84
DETECTIVE DNA
accurate diagnosis would be only made if that flanking region or the 'marker' is present close to the defective gene. This is because a gene very close to a known marker segment is coinherited from one generation to another as chances of recombination resulting in their separation are extremely few in a given family. Thus, a closely linked marker is inherited the same way as the gene. For finding out markers closely linked to a particular defective gene, DNA analysis of several individuals, both affected and unaffected by that disorder but all belonging to a single, large family is done. The method is basically of 'hit & trial'. In this a variety of restriction enzymes are used for cutting the DNA and several probes, each designed to identify a particular region of that gene are tested. The resulting RFLP patterns obtained are then studied for identifying a
UNA patterns of four patterns
DNA patterns of four normal persons
Band common to patient and normal Band varies in all Band common to samples patients
persons
Band common to normal
people
If a site of a restriction enzyme is altered in a DNA segment present close to a defective gene, it acts as a marker for detecting that genetic defect
85
closely linked marker that is coinherited with the normal or the mutated gene. For example, a particular restriction site - may be found to be always present or always absent in D N A of the patients. The enzyme used in this case and the particular probe used for hunting out that marker are thus specified. The final proof of identifying the site of a particular genetic defect, however, comes only after amplifying that gene by either cloning or using PCR and studying its base sequence. For this, DNA sequencing of that region is done which albeit laborious, provides the ultimate answer for the occurrence of a genetic defect. Search for linked markers for several genetic defects is an ongoing research effort. Many genetic diseases can be today
Beta (P) chains
Heme
group
P globin peptide
Alpha (a) chains
Oxygen binding site
Even a tiny genetic defect could be fatal. Sickle cell anemia is the result of alteration of a single amino acid in beta globin chain of hemoglobin. The hemoglobin molecule (inset)
86
DETECTIVE DNA
Red blood cells in sickle cell anemia
diagnosed specifically based on distinguishable RFLP patterns of normal and defective genes. A classic example is of a genetic disorder called sickle cell anemia where the defect lies in the hemoglobin molecule a vital life sustaining protein. This protein, in adults, comprises four peptides, two alpha globin chains and two beta globin chains. Hemoglobin is a huge molecule made of about 300 basic units called amino acids. The difference in normal and mutated forms lies only in a single amino acid. The sole chemical difference is that the amino acid 'glutamine' present at the sixth position of the beta globin chain is replaced by 'valine' in the abnormal hemoglobin molecule. This tiny error leads to such a fatal disease as the mutated hemoglobin cannot carry the required amount of oxygen from lungs to the various body parts. The
87
state is of acute anemia. The red blood cells which carry the hemoglobin assume the characteristic sickled shape. It was Vernon M. I N G R A M of the Cambridge University who traced the molecular origin of sickle cell anemia. The point mutation which is a single base substitution occurs in the sixth codon of the beta globin gene. This changes the sequence of the codon GAG to GTG. So instead of the base 'adenine' which is normally present at that site in beta globin gene, thymine is present. The sickle cell trait is, however, expressed only in those patients who inherit two defective genes, one from each parent. Thus, it is an autosomal recessive disorder. Sickle cell anemia is one of the first genetic disorders having a definitive diagnosis using RFLP analysis. This is so
Carrier father
Carrier mother
N n
N ni
N N
Normal
N n
Carrier Carrier I
N n
n n
Affected Normal gene (N) Faulty gene (n)
One chance in four Inheritance of autosomal recessive disorders
88
DETECTIVE DNA
because the mutation, by chance, happens to be present in the recognition site of two restriction enzymes: Dde I and Mst II. The mutation in beta globin gene causing sickle eel! anemia eliminates the restriction site of the enzyme Dde I. Thus, in affected individuals, the mutant DNA gives a single large band as no cleavage occurs at the Dde I site, which is absent. The distinct fragment confirming the diagnosis of sickle cell anemia is 376 base pairs long. In normal DNA, Dde I cuts at its site that is, -CTNAG (N means any nucleotide), producing two smaller fragments of 201 and 175 base pairs respectively. Using a radioactive detective having sequence complementary to this region of beta globin gene, distinct RFLP patterns are obtained for normal and faulty genes.
Normal p globin gene
DNA with sickle mutation in p globin gene Dde I cleavage
Electrophoresis
and blot
201 bp 175 bp
376 base pairs (bp)
Using the restriction enzyme, Dde I, a distinct DNA pattern helps in diagnosis of sickle cell anemia
89
The e n z y m e , Mst II which recognizes the sequence: C C T N A G G also does not cleave the D N A in affected individuals as the mutation occurs here changing its recognition sequence. Consequently, a homozygous sickle cell patient, having two faulty genes, produces a single long fragment of 1350 base pairs. Mst II cuts the DNA of normal persons into a distinct 1150 base pairs long fragment and a smaller fragment of 200 base pairs. The RFLP pattern of a carrier or heterozygous individual shows both the long and the short fragments as that person inherits one normal and one faulty gene. These carriers are asymptomatic as they are not affected themselves but can pass the trait to their offsprings. The specific probe used in this analysis is the 1150 base pairs long, Mst II fragment of the beta globin gene. Thus restriction fragments obtained on cutting D N A with above enzymes and probed with special detectives reveal a polymorphic pattern or variation in normal, carriers and affected persons. These RFLPs are detected as characteristic band patterns. A faulty gene can even be detected in an unborn child. For this, a sample of the fetal cells are first obtained usually by a technique called amniocentesis, where a small quantity of amniotic fluid is extracted from the womb at about 16-18 week of pregnancy. Fetal cells are then normally grown in an artificial medium to obtain sufficient quantity of D N A for the test. However, using PCR, fetal DNA can be amplified much faster. Prenatal diagnosis is pertinent in cases where there is a family history of a genetic disorder. The awareness that the child growing in the womb is destined to suffer from say, the fatal sickle cell trait, would alarm the couples of the lurking danger allowing them to take a timely decision to terminate that pregnancy. Most genetic disorders unlike sickle cell anemia are due to a variety of mutations involving several base sequences. One
90
DETECTIVE DNA Mst II cuts normal P^globin gene at three sites
1150 bp
200 bp
CCTNAGG
CCTGAGG
CCTNAGG
Detective spans the altered DNA
CCTNAGG
CCTGTGG
CCTNAGG
Mst II site is lost by sickle cell mutation
Southern blot
Characteristic band patterns of Mst II cut DNA of parents who are carriers of sickle cell trait, a normal offspring and an affected fetus. Of the two DNA fragments, the 1350 bp fragment is associated with the defective gene
91
Amniotic fluid Fetus
Centrifuged Syringe
Fluid
Uterus
Fetal cells I cultured
Chromosomes
examined
Is the fetus growing in the womb normal? The technique, amniocentesis helps to find it out
such disorder again affecting the globin genes is thalassemia. Described by C O O L E Y in 1925, this disorder is characterized by reduced synthesis of either alpha or beta globin chains of the hemoglobin molecule. This results in an excess of the other chain. The excess chains are unstable which precipitate causing acute anemia, iron overload and ineffective synthesis of red blood cells. This defect is targeted on the globin genes that dish out the alpha and beta globin molecules. In alpha thalassemia, there is a deletion of some nucleotide sequences in the alpha globin gene of the patient and in beta thalassemia
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DETECTIVE DNA
the mutation lies in the beta globin gene. These mutations can be easily detected if, by chance, the mutated gene is linked to a restriction enzyme site that is absent in the normal D N A or vice versa. Detection of these closely linked markers would indicate the inheritance of that gene too. However, in absence of a convenient restriction site, the sequence of mutation should be known so that a probe having a sequence complementary to the mutant gene can be synthesized. Obvious variations in RFLP patterns would then help in diagnosing the carriers and normal individuals from the victims of thalassemia major which are carrying two defective genes. Thalassemia is also an autosomal recessive blood disorder. The carriers suffer from a milder version of the disorder, sometimes undetectable, called thalassemia minor. Genetic studies have demonstrated as many as 28 mutated points on the globin genes of thalassemia patients. Another most frequent fatal genetic disorder, spread all over Europe is called cystic fibrosis. Specific mutations have been identified at several points in the cystic fibrosis transport regulator (CFTR) gene. The carrier frequency of these mutations in Caucasians is about one in 25. The CFTR gene was isolated in 1989 by Lap-Chee TSUI of the Hospital for Sick Children, Toronto and Francis COLLINS of the University of Michigan. It is located on chromosome 7. As the disorder is autosomal recessive, a child suffers from cystic fibrosis only if both the parents have passed on a defective gene. The protein coded by the CFTR gene forms channels in cell membranes for transport of chloride ions across the walls of cells mainly lining the intestines and air passages. A defective protein prevents just that. This leads to increased salt content of sweat of the victims besides changes in the secretions of a gland called pancreas. A thick, sticky mucous thus clogs the lungs and gut resulting in respiratory problems. Many patients have shown a three base pair deletion in the CFTR gene. Specific diagnosis of this disorder is possible by RFLP analysis.
93
Chromosome 7
Nucleotide sequence in CFTR gene AT cA T
Isoleucine
Isoleucine Deleted in many patients with cystic fibrosis
c" _r T GG
Phenylalanine
CFTR gene
Glycine
T
Valine
GT T,
Cursed by one's own DNA: A deletion of three nucleotides from the CFTR gene causes cystic fibrosis. Lap-Chee Tsui (inset)
A second kind of genetic disorders identifiable by variations in restriction patterns of DNA are autosomal dominant disorders. In such disorders, a single copy of the faulty gene inherited from either parent is sufficient to spell disaster. Individuals with two copies of the defective gene show the same degree of illness. Huntington's disease is one such disease afflicting mainly the Americans. This diseases is
94 Carbohydrate Site of common phenylalanine deletion
DETECTIVE DNA
Cell membrane
ATP Niiclcotid^^L binding ilotwm| Cytoplasm Regulatory domain Normal lung Normal CFTR Channel Chloride Phosphate
ATP Chloride Nucleotide binding domain
Lung affected by cystic fibrosis Defective CFTR channel Chloride
Sodium
Sodium Airway
Cells lining the air passage In cystic fibrosis, cells lining the air passage prevent chloride m o v e m e n t causing enhanced uptake of sodium. A n intact C F T R protein present in the outer m e m b r a n e of a cell (inset) n a m e d after G e o r g e H U N T I N G T O N , an A m e r i c a n Physician w h o first d e s c r i b e d t h e i n h e r i t e d n a t u r e of this d i s e a s e . It is a degenerative, age-onset disorder of the brain that results in loss of b o d y ' s response to v a r i o u s sensations a n d total m e n t a l
95
Faulty gene (D) Normal gene (d)
Affected father
Normal mother
D d.
d d -
Dd
Dd
d d
Normal Affected Normal
Affected
A fifty-fifty chance Inheritance of autosomal dominant disorders. George Huntington (inset) was the first to describe dominant inheritance of Huntington chorea, a disease named after him
incapacitation. The disease appears late in life, only after child bearing years. The faulty, culprit gene causing this disorder was identified in 1983 by scientists led by James GUSELLA, a Harvard molecular geneticist. A marker DNA sequence linked to the faulty gene i n Huntington's disorder was identified by Gusella using a special detective. The diagnostic test is now being done at the Massachusetts General
96
DETECTIVE DNA
A time bomb ticking in the genes? Such is the terror of the age-onset Huntington's disease. James Gusella (inset) discovered the Huntington's disease gene
97
Hospital in Boston and the John Hopkins Hospital in Baltimore. In an interesting study, Nancy WEXLER, a psychologist at the Columbia University and President of the Hereditary Disease Foundation has traced the inheritance of this disease in the world's largest family of Huntingtons' victims composed of 9000 members, at lake Maracaibo, Venezuela. Residing on chromosome 4, the defective gene has been linked to a repeat sequence comprising three nucleotides namely,
A 'water village' on Lake Maracaibo, Venezuela where lives the largest family of persons suffering from Huntington's disease. Nancy Wexler (inset) developed a diagnostic test for this disease
98
DETECTIVE DNA
CAG. These trinucleotide repeats are formed as a result of mutations that occur during DNA copying. The insertion of extra nucleotides increases the repeat number. However, a minimum number of 36 C A G repeats is the threshold length supposed to cause the disease. This linked marker for the faulty gene acts like a flag for tracking the defect through successive generations of a family. Unfortunately, Wexler, one of the top researchers in this field too carries this ill-fated gene! A third kind of genetic disorder is X-linked which mainly affects males. Women are carriers if one of their two X-chromosomes bears the genetic defect. Whereas men suffering from it have the defect in their single X chromosome. A classic example of this disorder is Duchennes muscular dystrophy
A defective gene on an X-chromosome of mother
Carrier mother
Normal father
XX
XY
XY XY XX
Affected son Normal son Normal daughter Carrier daughter
X X
Males at risk Inheritance of X-linked disorders
99
The Boston Children's Hospital team that found the Duchenne muscular dystrophy (DMD) gene. Left to right: Eric Hoffman, Michel Koenig, Chris Feener, Marybeth Mc Afee, Corlee Bertelson and team leader Louis Kunkel
(DMD). The affected child shows delay and difficulty in walking. As the muscles grow weak, the victim is confined to wheel chairs. Further complications arise as muscles that control breathing weaken. The disease is due to lack of a protein called 'dystrophin' which is due to the faulty dystrophin gene. This gene is located on the X chromosome. It was discovered in 1986 by a team of scientists led by Louis K U N K E L at the Boston Children's Hospital. Mutations resulting in deletion of several nucleotide sequences have been shown to occur in the faulty gene. Prenatal diagnosis of this disorder by detecting the deletions in the dystrophin gene in a foetus has become possible today. Genetic conselling enabling selective timely abortion is so far the only way of reducing the incidence of this disease. Besides several genetic disorders, early detection of some cancers by RFLP analysis has also become possible. In most
100
DETECTIVE DNA
c a s e s , the normal genes con- Scaly skin (Ichthyosis) trolling cell growth and dif~Albinism of the eye ferentiation un^Duchenne muscular dystrophy dergo mutations Retinitis pigmentosa which convert them to cancer causing genes called onco" A form of hemolytic anemia genes. Even a change in a sin+ Cleft palate gle base pair m a y , in s o m e cases, b e suffi.. Gout cient to arouse j . Lesch-Nyhan Syndrome the cancer monHemophilia B ster. For detecting these ____ - Fragile X mental retardation mutations, usuI Manic depressive illness, Colourblindness, J Hemophilia A, Diabetes incipidus ally PCR amplified region of Human diseases associated with defective genes that oncogene is present on the X-chromosome analyzed using specially designed detectives. In one such study, Johannes BOS and his team at the State University of Leiden, Netherlands have shown that more than a third of human colon cancers carry mutations in the 'ras' oncogene. Diagnosis of a type of blood cancer, chronic myeloid leukemia by RFLP analysis is yet another revolutionary step towards DNA diagnostics. Earlier, diagnosis of this cancer depended only on the identification of an abnormal chromosome called the 'Philadelphia' (Ph 1 ) chromosome. This chromosome is a chimera formed by a translocation that fuses a fragment of chromosome 9 with chromosome 22. N o w using
101
specially designed detectives, it is possible to identify the markers or certain known DNA segments which are specific for the Ph 1 chromosome. Also, this test is done after t h e b o n e marrow transplant in leukemia pat i e n t s . If t h e transplant is s u c c e s s f u l , the RFLP pattern erf
1 > -sl'tl. < V
^ ?H. >
w am
mm
the p a t i e n t ' s DNA diagnosis of chronic myeloid leukemia. DNA DNA w o u l d profile of normal individuals lack both the DNA also s h o w the fragments specific for this defect
donor's normal profile. If not so, the transplant has been rejected. Tracking down of molecular changes responsible for the development of several other cancers is underway. Specific mutations in a tumour suppressor gene called p53 have been identified in persons having hereditary predisposition to breast cancer. By genetic analysis the possibility of knowing the susceptibility of a person to an autoimmune disorder is also in the offing. These disorders are caused by an abnormal attack of the defence machinery on the body's own tissues. Having a genetic component, these disorders have been found to be associated with specific alterations in certain regions of the blueprint called the histocompatibility complex that com-
102
DETECTIVE DNA
prises several genes. These genes code for special molecules that play a major role in the normal functioning of the body's defence system. Tracing of specific markers linked to a gene of this complex responsible for a particular autoimmune disorder is underway. Man's unsatiable urge to know more and more about the code of life has culminated in a commendable scientific venture o f this century the Human Genome Project (HGP). It aims to identify and decode the entire information buried
103
inside the blueprint of man. Unquestionably, it would be a great scientific accomplishment. The entire genetic report card of a person could be then revealed bringing to light all the good and bad genes it contains. But there is the other side of the coin too. With this knowledge, persons predisposed to a genetic disorder would become known. Stigmatized, such persons would suffer the brunt of the hi-tech as doors to job opportunities or medical insurances might shut for them. As one forsees, life of persons carrying an ill-fated gene would be miserable. Perhaps, this would usher in a new era of selecting 'good genes' in the human population. A controlled use of the new found information is, therefore, of utmost importance. Mapping of diseased genes has led to gene therapy which is basically the doctoring of the abnormal genetic sequences by incorporating the normal gene in place of the mutated counterpart. Several genetic diseases, therefore, await a cure. But all this owes a great deal to the key player, the D N A detective that has actually brought this revolution in disease diagnosis.
M
Catching the Culprit: A Microbe
icrobial diseases are a constant threat and they would remain so as long as microbes exist. But, thanks to drugs, the burden of these illnesses could be eased. Many diseases caused by hostile organisms called parasites, are completely curable. When they occur they can be treated and cured, provided the disease is first correctly diagnosed. The diagnosis of a particular disease is, in fact, a tough job and requires considerable skill especially for detecting those diseases which lack distinctive symptoms. The usual method for identifying a parasitic disease is by examination of the patient's blood, sputum or urine samples under the microscope for the presence of the parasite. This however, requires the isolation of the parasite from pathological specimens, and culturing them which at times is a time-consuming process since some parasitic forms grow extremely slow in cultures. Moreover, the characteristic appearance of a parasite of one strain may be indistinguishable from that of another, thus making it difficult and sometimes impossible to identify a parasite. These difficulties can lead to a delayed diagnosis or as happens more often, to a wrong diagnosis
CATCHING THE CULPRIT: A MICROBE
105
of a disease. The ultimate result of this could cost the patient his life. However, with the advent of DNA profiling technology, the unique genetic profile of a particular pathogen can be known, thus detecting it much before the appearance of the disease symptoms. A preventive prophylactic treatment, if initiated, would then kill the pathogen in many non-symptomatic but potential patients. The sensitive molecular tool having an eye for discriminating closely related organisms is the detective D N A specially designed for detecting a particular species of a microbe. Thus D N A probes used for the identification of the pathogen are specific D N A sequences of the parasite's genetic material. These are sequences which are unique to the parasite and even the most closely related species and strains are devoid of those sequences. Such a species-specific D N A fragment of the parasite is first identified from among the many differences that exist between the parasite and its relatives. This is done by using the same techniques of D N A hybridization among the D N A of the patho'gen and of an array of its close relatives. A D N A sequence that is seen only in the pathogen and not in any of its relatives is selected. The procedure which is mostly a hit and trial method, is a very important Step because without identification of a unique sequence, a probe D N A cannot be prepared. So, for diagnosing a parasitic disease, the parasite is first isolated from tissue samples of infected persons followed by extraction of D N A from parasites. The D N A is then cut with an appropriate restriction enzyme, size sorted by gel electrophoresis and Southern blotted. It is subsequently hybridized with the special detective whose sequence is complementary to a D N A fragment 'unique' to the pathogen being detected. Appearance of a dark band on the X-ray film means 'yes', that the particular bug tested for is present in the tissue sample. The absence of the dark band obviously proves that the
106
DETECTIVE DNA
Patient
Unique DNA fragment
of the bug Infected tissue sample
Isolation of the bug
Bug's DNA is chopped
>(J
DNA probe binds to unique fragment of the bug V _ r< j i; film
Gel electrophoresis
Southern blotting
Bands
invisible
E m p l o y i n g a D N A d e t e c t i v e to identify a d i s e a s e - c a u s i n g b u g
107
parasite against which the probe was designed, is absent. Results of some such efforts are encouraging. In the diagnosis of parasitic infections caused by viruses, bacterial or protozoan species preliminary trials have proved the singular ability of D N A probes to detect the concerned pathogens. Besides the single copy unique DNA sequences of a bug, the basis of such probes is the presence of substantial amount of D N A in an organism that does not code for any proteins, but rather consists of moderately or highly repeated sequence clusters. These repetitive sequences make especially suitable D N A probes for distinguishing even closely related organisms. This is so, because, unlike protein coding D N A sequences which are almost the same in many related species, these sequences tend to differ among different species and sub-species. A sequence unique to a particular parasite type will precisely identify that type. Moreover, due to high copy number, the sensitivity of these pathogen-specific hybridization tests is very high, detecting down a single bug of that species or sub-species.
108
DETECTIVE DNA
Many disease-causing organisms that include bacteria, viruses, fungi and protozoan parasites can be specifically detected once DNA fragments specific to that organism are known. In the light of PCR, the sensitivity of detective D N A could be further enhanced. PCR amplified D N A of the pathogen can even be directly probed with a special detective that identifies a species-specific DNA fragment of a particular pathogen. This would bypass the steps of cutting DNA, running it on gel and Southern blotting. Thus, instead of a unique RFLP pattern of the bug's DNA, identification would be on the basis of dark spots or regions on the X-ray film that indicate the binding of the detective to its target.
DNA seen under UV light
DNA extracted DNA fragment to be amplified PCR Gel electrophoresis
Analysing the PCR amplified DNA
Specific probes for the detection of many parasitic diseases have been designed, of which some have successfully passed the experimental laboratory trials. One such disease is tuberculosis (TB) the scourge of mankind particularly those of the Third World. Despite the availability of highly effective drugs, TB still continues to cause devastating illness. This is attributed to the so far lack of foolproof, rapid and early diagnosis of the disease. Conventional methods of its diagnosis based on smear staining and radiological findings are unreliable since a wide spectrum of diseases of the lungs
109
simulate symptoms of TB and are therefore, indistinguishable from TB. As the TB bug takes several weeks to grow in a culture medium, these methods are time consuming. Therefore, it is not surprising that worldwide about 3 million people die of this disease every year. The formidable challenge of a definite diagnosis of TB has at last been successfully met due to this D N A analysis technique. Specific D N A fragments of the T B bug, Mycobacterium tuberculosis used as probes can unequivocally identify the parasite even in clinical samples which do not show these pathogens under the microscope. In India much work is being done for identifying such species-specific fragments of the TB bug. In one such attempt, Jaya S. TYAGI of the Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi has developed a PCR-based non-radioactive test for detection of microorganisms of the genus, Mycobacterium in uncultured clinical specimens. The primers for this PCR are so specific for mycobacteria that they amplify only a particular 'unique' DNA sequence of this bacterium which confirms its identity. The amplified D N A is electrophoresed on agarose gel and visualized by e t h i d i u m b r o m i d e staining. This rapid screening test is not only genus-specific and speedy but also highly sensitive as it detects down even a single Mycobacterium. Tyagi's group has also identified a unique single copy DNA sequence in the genetic material of the TB bug. This portion of the bug's D N A is selectively amplified using specific primers in a PCR assay and detected by a non-radioactive method. Definitive detection of the TB
Jaya S. Tyagi is working on devising a DNA diagnostic test for TB
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DETECTIVE DNA
DNA probe
Hydroxy apatite
Bug's DNA
Quick hybridization
Hybrids separated
Hybrids
detected
The Gen-Probe technology for molecular diagnosis of TB
bug in clinical samples using this PCR assay has been shown. Based on DNA hybridization, a complete testing system for TB is now being marketed by Gen-Probe Inc., a Californian company. The test is very simple. In this, the radiolab e l e d probe DNA is mixed with the DNA of the organism in a test solution. The probe DNA molecules on colliding with the complementary target sequences stick to them and form hybrids. Separation of the bound and the free probe is then carried out by immobilizing the hybrids onto a chemical matrix, made of hydroxyapatite. The excess probe is washed away and radioactivity of the bound probe is measured using a special instrument called gamma counter. Similarly, efforts are in full swing for designing strain specific DNA detectives for the diagnosis of leprosy another major mycobacterial disease affecting mostly the third world. Specific stretches of the genetic material of the bug causing leprosy, namely Mycobacterium leprae are now known which are potentially useful for identifying this bug in tissues of leprosy patients. The credit for this work goes to a team of
111
s c i e n t i s t s led b y I n d i r a NATH, Professor and Head, D e p a r t m e n t of B i o t e c h n o logy, AIIMS, N e w Delhi who has developed a PCR assay for detecting the leprosy bug in clinical samples. The genetic material of M.leprae used to develop this PCR assay actually encodes a protein which elicits the production of molecules', called antibodies, in the victim's body. Based on the n u c l e o t i d e s e q u e n c e of the gene coding this antigen,
p r i m e r s for the P C R assay
Indira Nath A pioneer of Indira Nath A pioneer of leprosy research in India leprosy research in India
were made. In this assay the primers bind to the complementary regions of the bug's DNA and amplify them manifold. The so amplified DNA is then visualized by running it on agarose gel and staining with ethidium bromide. This test has been found to be both sensitive and specific with a detection level of as low as 10 bacilli. This PCR assay also helps to differentiate M. leprae from a variety of other bacteria, including closely related mycobacterial species, thus confirming the identity of the leprosy bug where conventional techniques seem inadequate. In yet another similar attempt, a team of Indian scientists led by P. Usha S A R M A at the Centre for Biochemical Technology (CBT), N e w Delhi, a premier CSIR laboratory have developed a PCR assay for definitive diagnosis of aspergillosis a fungal disease. The causative fungus, Aspergillus fumigatus mainly invades the lung tissues. As symptoms of aspergillosis simulate those of TB and pneumonia, the hapless victim is deprived of proper and timely medication causing irreversible lung damage. This opportunistic fungus particularly invades persons with impaired defence machinery such
112
DETECTIVE DNA
as those suffering from AIDS (Acquired Immuno Deficiency Syndrome), TB and post-transplant c a s e s - w h o are susceptible to a host of infections. Rapid identification of A. fumigatus at an early stage of infection is today possible, thanks to Sarma's team. P. Usha Sarma has made the molecular detection CBT scienA computer generated model of a portion tists h a v e idenof a major antigen of this fungus (inset) tified a protein of the fungus, an antigen which elicits a strong i m m u n e reaction in the victim's body. Aportion of this fungal antigen w a s further analysed for its molecular composition, that is, the sequence of its amino acids the basic building blocks of any protein or peptide. From this peptide sequence, the stretch of genetic material which coded it was found. Based on this nucleotide sequence, primers for a.PCR assay were developed. Results of this PCR assay are encouraging as specific identification of the bug, A. fumigatus is possible even in clinical samples. For isolation of D N A from the fungus present in clinical samples, C B T scientists h a v e developed a novel method that uses chitinase and microw a v e treatment. D N A hybridization probes have also been developed for some of the most widespread protozoan diseases. Control of sleeping sickness, one of the most devastating diseases of the
of the fungus, Aspergillus fumigatus possible.
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Trypanosomes Blood parasites that cause African sleeping sickness
tropical belt, also seems to be in sight. D N A probes for the identification of the causative protozoan bug: Trypanosoma, have been developed at the International Laboratory for Research on Animal Diseases (ILRAD), Kenya. Since the sequences that make up these probes are present in high copy number ifi the genomes of trypanosome populations, these sequences have helped in developing highly sensitive hybridization tests. Further, the utility of D N A probes has also been extended to the detection of infected insect vectors and intermediate hosts which carry some stages of the parasite. Trypanosomes have been detected in infected tsetse flies by this method at least in the laboratory specimens squashed directly on the nitrocellulose filter. Similarly, Kala-azar a protozoan disease marked by a variety of skin lesions could now be controlled. DNA probes which can specifically identify the parasite, Leishmania dono-
124 DETECTIVE DNA
vani from lesions, sandfly vectors and the intermediate mammalian reservoirs have been successfully developed. DNA probes for the diagnosis of malaria, a prevalent disease of the tropics, are also in the offing. The Plasmodium parasite is transmitted by various species of the anopheline mosquito to the human host. The World Health Organization has estimated that more than 40 percent of the world's population is at risk for malaria infection. DNA probes specific for the parasite have been demonstrated to detect the infection directly in finger-prick blood of infected patients. For an early and effective cure of gastro-intestinal diseases, prompt identification of the parasite is the key factor. A major cause of death in our country, these diseases are known to be caused by several viruses, bacteria and protozoan parasites. Growing the pathogen for detecting is a tedious job. DNA probes are proving to be a unique tool for specific identification of each of these bugs. Using special detectives it is possible to detect several viruses, particularly the AIDS virus called HIV (Human Immuno Deficiency virus). Unlike other viruses, HIV has a peculiar way of infecting its host. It first converts its genetic material from RNA to DNA. The viral DNA then incorporates in the host genetic material where it sits dormant for an unpredictable period, ranging from a few months to several years. Normally, HIV is detected by the presence of specific antibodies in blood which the host body produces in response to this infection. However, detecting AIDS directly based on hybridization of special detectives with specific portions of the viral genetic material is now possible. This would enable to estimate the virus burden, a symptom-free person carries. Higher the burden, greater would be the risk of coming down with AIDS. An early drug therapv can be started in such persons. Several cancers have also been associated with certain viruses like Epstein-Barr virus, human papilloma virus and
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A computer generated picture of human immuno deficiency virus (HIV) the virus that causes AIDS
hepatitis B virus. Specific detection of these viruses is in offing too. However, for making diagnosis of microbial diseases truly useful, laboratories worldwide should be able to perform these tests. Also, the tests should be reproducible in large number of samples and be adaptable to field trials. In this light, the focus nowadays is on developing techniques for enzymatic detection of the target DNA. Scientists are increasingly making biotinylated probes for identifying particular species of disease-causing microorganisms. Hybridization, in this case, is detected by adding streptavidin- enzyme complex as streptavidin unmistakenly binds just to biotin. A coloured product is formed on adding an appropriate substrate for the enzyme used. Detectives with a tag of a chemiluminiscent substance or a fluorescent dye are also being developed. The enzyme
116
DETECTIVE DNA
Tuciferase' that is responsible for the glow of fireflies is one such candidate suitable for tagging onto the detective DNA. In presence of adenosine triphosphate (ATP), the energy currency of a cell, and the substrate Tuciferin', light is emitted which can be visualized or recorded on a photographic film. It is indeed a safer and cheaper substrate to radiolabeled probes. Diagnostic kits, based on chemiluminiscent probes, for detection of Hepatitis-B virus and Epstein Barr virus among others are commercially available in USA. Similarly, some fluorescent dyes for labelling the detective have been developed by the Department of Forensic Science Service, UK. In yet another breakthrough, a target D N A can be quickly detected by a simple test tube method. The credit goes to Andrea G A R M A N of Zeneca Pharmaceuticals, Macclesfield and Peter SAMMES and his team at Brunei University, Middlesex, UK. Instead of a radioactive molecule as tag, scientists attached an ion of europium, an element than can luminesce. The snag, however, was that europium does not radiate light when surrounded by hydroxyl ions present in the test soluTarget DNA sequence Sensitizer molecule Sensitizer "switches on' europium ion
Probe DNA
Europium ion
Europium ion luminesces
The test tube detective : A simple and rapid test for analyzing DNA
117
tion. The scientific genius of Garman and Sammes met this challenge by designing a second reagent consisting of a short organic molecule. The latter acted as a 'sensitizer' for europium. This molecule has a 'head' and a 'tail', each capable of performing a different job. The head of the sensitizer binds to the europium ion in the probe. The tail, on the other hand, attaches to the double-stranded D N A formed after the probe binds to its target DNA. So, as both head and tail are attached to each other, they form a 'bridge' complex. It is this structure which has the effect of driving away the hydroxyl ions around europium which thus luminesces. This simple D N A detection test having far-reaching practical uses would surely prove to be immensely vital in catching the culprit a microbe. The quick and reliable D N A analytical techniques are slowly but steadily replacing the traditional methods of disease diagnosis. A prompt, effective patient therapy would further control the diseases. No wonder the detective is now eagerly looked forward to come into wide use by disease control workers and epidemiologists.
he tremendous impact of DNA diagnostic techiques is being felt in the food industry as well. Specially designed detectives are being increasingly employed for the detection of food borne pathogens and diagnosis of crop and livestock diseases. These detectives have added an exciting new dimension to plant and animal breeding besides authenticity testing of meat products and plant materials.
A Boon to Food Industry
The diagnostic performance of this genetic tool is unparalleled. It clearly reveals the presence of any specific target D N A sequence based on hybridization of complementary sequences, and thus, the presence of an organism containing that sequence. Keeping in view its extensive utility, the food science community has rather promptly adopted this technology for commercial application. Detection of various food borne and water borne human pathogens is now possible. This includes the identification of bugs that cause typhoid, cholera, intestinal inflammation and even viruses like those that cause hepatitis and influenza. The detective, in each case, selectively identifies a particular organism
A BOON TO FOOD INDUSTRY
119
since they are tailored to be strain-specific. Detection of different strains of yeast is another major development. Yeasts are an important group of microorganisms whose numerous strains, with special characYeast is the major workhorse for making fermented food products teristics, are being used in the commercial production of beers, wines and bakery items. However, certain strains are known to spoil a vast range of food materials. For accurate identification of a yeast strain, stretches of D N A unique to that strain are first identified. Normally, these DNA sequences are radiolabeled and used as a detective for identifying that particular yeast strain. However, in another method, regions of these strain-specific DNA sequences are used as a primer in PCR, thus amplifying only these segments of a yeast sample being tested. Amplification of these strain-specific sequences will occur only if they are present in the test solution. In other words, amplification means detection of that particular species/strain. The amplified DNA is visualized by gel electrophoresis followed by staining with ethidium bromide. Amplified DNA can be seen as a thick shining band under UV light. Several common food spoiling yeasts can be detected in this way. The hybridization format commonly used for detecting food borne pathogens are dot blot analysis and colony hybridization. In colony hybridizaton, several food samples can be tested together for the presence of a particular species of
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DETECTIVE DNA
Nitrocellulose
filter
Master plate
Bacterial DNA trapped in the filter is unwound
A replica of bacterial colonies on the filter
Probe binds to complementary DNA
Filter exposed to X-ray film
Bacterial colony to which the probe has bound
Colony hybridization
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pathogen. The bacterial strains contained in the food samples are made to grow in a culture medium, usually Agar. Bacteria of each strain grows and forms a colony of identical cells which looks like a rounded structure on the growth medium. These colonies of cells are blotted onto a filter and lysed directly on it by using certain chemicals. DNA contained in the cells firmly adheres to the filter while the cell debris is washed away. By alkali treatment, the double strands of DNA are reduced to single strands. Special probes which specifically identify a particular species of the pathogen are now
X-ray film
Exposed region
Nitrocellulose
filter!
Colony
hybridization
Radioactive
DNA
Filter with bacterial colonies
Dark spot corresponds to a particular bacterial colony A p p e a r a n c e o f d a r k spots on the X-ray film c o r r e s p o n d to bacterial colonies to which the radioactive p r o b e has b o u n d
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DETECTIVE DNA
made to hybridize with DNA of the pathogen lying immobilized on the filter. On exposing this filter to an X-ray film in dark for a defined period, followed by developing it, dark spots appear where the detective has bound to the pathogen's DNA. Thus, the culprit is detected from amongst several closely related species. Colony hybridization identifies a particular species of a pathogen in mixed cultures and it also facilitates the isolation of pure culture of that bacterial strain. Raw materials that enter a food industry and the finished products that are subsequently released to the market could be thus tested for such pathogens. Clearly, this would reduce the outbreaks of food borne diseases. What's more, non-pathogenic food-related organisms that are economically important can also be detected by special detectives. One such organism is Lactobacillus curvatus which ferments meat. It can be specifically identified from amongst a large number of Lactobacillus species and then used in the manufacture of fermented meat products.
Lactobacillus A friendly bacterium
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Notwithstanding the advances made in protecting plants and animals against various diseases, ineffective disease control of crops and livestock results in heavy loss to the food industry every year. However, with the advent of DNA hybridization technology, accurate and rapid identification of a pathogen has become feasible as soon as it enters the host body. Therefore, even before the onset of disease, quick therapeutic measures against the disease can be taken. Special detectives which accurately identify a particular crop or livestock pathogen have been designed. For example, several insect pests can now be distinguished from one another by comparing the unique patterns of their D N A profiles. This would help in early detection of the outbreak of pests thus enabling to eradicate them before the crop is destroyed. L one study, J. B E N N E T and N.Z. E H T E S H A M at the Plant molecular biology unit of International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi are studying the variations in DNA of different varieties of rice pests called the rice gall midges. Selective breeding of plants and animals is yet another area where detective D N A is immensely useful. This would replace the laborious, expensive and time-consuming traditional practice of selecting appropriate organisms, crossing them and then selecting the most suitable progeny for further propagation. The detectives are exploited to detect specific D N A sequences that behave as molecular markers. Breeders use these markers as guides for the selection of appropriate organisms. In an interesting study, Sher ALI of National Institute of Immunology (Nil), New Delhi, has developed probes for genetic analysis of different breeds of buffalo. This work is poised to be immensely useful in selecting appropriate breeds of the animal as mating partners for propagation of the desired genetic traits. Such pedigree analysis of livestock and wild animals would also help in understanding their evolutionary relationship with other related species.
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DETECTIVE DNA
Kb
->23.1
9.4
6.5
4.4
2.3
Pedigree analysis at molecular level: Buffalo DNA suitably cut and hybridized with a special radioactive probe produces multi-locus bands. Each band of the offsprings DNA profiles (Lanes o) can be traced back to that of their respective mother (Lanes O) and father (Lane ) (Courtesy of Dr Sher Ali, Nil, New Delhi)
Breeding of unrelated species can also be encouraged as it would bring variations in offsprings. Inbreeding among endangered species can be, similarly avoided, as weaker animals are produced. Controlled breeding of rare species can be achieved. Such breeding strategies aimed at conservation
125
of rare plants and animals, and breeding of their diseaseresistant varieties is proving a boon to the food industry. D N A detectives have also lend themselves to certain food related authentication problems, such as the identification of raw or cooked meat from any species. Similarly, recognition of genetic fingerprints of plant varieties would help in assessing distinctness, uniformity and stability of seed samples. Authentication of labels and documents is likely to be achieved by using a pen containing a solution of short strands of synthetic DNA. Marks of such a pen on a package can only be visualized by hybridization with a detective of complementary sequence. This would be indeed a unique way of marking a product. D N A detectives, the powerful diagnostic tools, have thus a rich store of promises for the food industry as they are poised for tackling its diverse problems in the coming years.
Hurdles to Hightech
eedless to say, DNA profiling has revolutionized the field of diagnostics, be it concerned with forensic identification, paternity testing, disease diagnosis or in food industry. However, there is a note of caution, that is, the biotechniques involved in this technology should be conducted meticulously and with utmost care. Any flaw therein can produce incorrect results that may lead to disastrous consequences. Controversy over this technology, therefore, arose when it was realized that in criminal cases if the D N A profile of a suspect finds a match with a sample found at the site of crime, it may prove to be a sufficient warrant to convict that person.
However, in USA when a new scientific test is used as evidence in a court, it must first meet the so called 'Frye standard'. According to this, the judge must be convinced that the new technique is a fairly established one and has gained general acceptance in that particular field. This usually requires a pretrial hearing where no jury is present and lawyers from both sides, defence and prosecutor, argue the reliability of the new technique. Scientific experts in that field are also present for
128
DETECTIVE DNA
comments and any clarifications. It was the double murder case in Bronx in 1987 which, for the first time, thoroughly examined the reliability of DNA profiling in forensic identification although several trials based on this had been concluded earlier. By D N A profiling, Joseph Castro was nailed for the murder of a woman, Vilma Ponce and her two year old daughter as the blood stain found on his watch matched those of the victims. The tests confirming the identity of the killer, were conducted at Lifecodes Corporation. However, the scrutiny of the expert defence lawyers in pretrial hearings shrouded the DNA evidence in controversy. What's more is that some eminent biologists also considered the evidence from Lifecodes as unreliable. More such cases were to follow. O.J. Simpson, a ren o w n e d football player w h o murdered his ex-wife Nicoll Brown Simpson was caught for the tiny specks of his blood that were scraped off the pavement at the site of crime revealed his genetic signature. The tests were conducted at Cellmark Diagnostics. The reliability of D N A profiling w a s questioned by the defence experts in a m o v e to protect their client in pretrial hearings. The controversial arguments on the procedures used in collecting, labelling and testing the samples thus posed a hurdle to this hi-tech. Bogged down in arguments, this murder trial too could not b e concluded on D N A testing. Well, what are these hurdles to DNA profiling particularly when applied to forensic identification? Firstly, visual analysis of the bar code pattern of a DNA profile requires skill and expertise as it is not easy to interpret. Comparing two DNA profiles solely by eye can have errors. For example, a band corresponding to a D N A fragment containing 10 repeat sequences would be so close to a fragment with 11 such sequences that the two would be almost indistinguishable. So, a computer scanning device is needed to check and compare the DNA profiles by measuring accurately the exact size of each fragment that makes the bar code pattern. Secondly, in
HURDLES TO HIGH-TECH
129
criminal cases, when forensic samples are brought for the test, they are mostly contaminated like blood stains collected from a s i t e o f crime may be contaminated with bacteria. This may result in m i s l e a d i n g a p p a r e n t matches or mismatches. There may be a prese n c e of extra bands or s o m e bands may be m i s s i n g in a Interpreting the band pattern of DNA profiles is DN A profile. Bevery crucial sides microbes, other environmental factors like heat and moisture may degrade DNA. Therefore, collection of uncontaminated fresh samples is very important in DNA profiling. At every step of this analysis care should be taken in carrying out the technique meticulously. Since the test depends on a comparison of the length of different cut fragments, the DNA samples to be compared should be treated exactly the same way like cleaved with the same restriction enzyme, run on the same gel and probed with the same detective. Sometimes due to certain human errors, repeated DNA testing of a particular sample shows the same band
130
DETECTIVE DNA
X-ray film
Comparing two DNA profiles
=s
'
Bands match , , , Bands shtfted
Xtra
1 , , , Bands do not match
Bands match but

bands
present
For clearcut results, the biotechniques involved in DNA profiling should be performed carefully
pattern but the bands are out of alignment. The relative position of the bands, however, remains same. This problem is called 'band shift'. Any such tiny difference caused by sheer carelessness would lead to wrong results and misinterpretation thus defeating the purpose of separating guilt from innocence. The credibility of D N A profiling rests upon the statistical interpretation of the results. Calculating how likely it is for a match to occur by chance is extremely difficult. So far, computing the occurrence of a given DNA profile is based on calculating the probable frequency of each band in a given population. In other words, after measuring the size of D N A fragment of a particular band, the frequency of that fragment in a given population is determined by relying on population databases. For estimating the frequency of occurrence of a particular DNA profile, the individual frequencies of the various bands in that given population are multiplied. The snag here is, the assumption of random mating. Population databases are mainly based on the assumption that a DNA fragment of certain length is always evenly distributed throughout a large population group. This is however, a theoretical model of distribution of genes in a completely
HURDLES TO HIGH-TECH
131
mixed population. In reality, genes are usually not well shuffled in a large population. This is so because, there are subgroups within every population. As random mixing is not allowed in many such subpopulations, matings occur only within that group. This results in 'pooling' of certain characteristics in that subpopulation group. Thus a genetic trait becomes more common over a period of time in these isolated groups or subpopulations just as some traditions or customs are passed down several generations of a family. Clearly, there would be D N A fragments of a particular size which must be appearing more often within that subgroup. So it is possible that a repeat segment of a particular length may b e present in a member of the suspect's family. This only means that the chance of a match of a D N A fragment of certain length in a D N A profile should be considered in accordance to its frequency of occurrence in the subgroup of population to which that person belongs. This would give a more authentic picture of the chances of two individuals sharing the same DNA fragment. For example, extensive similarities are likely to occur in racial or ethnic groups as certain genetic traits are more common in them than in a random population. Therefore, ideally, separate database on gene frequencies for each subgroup of a given population should be available. All what is needed at this stage is that forensic science should be regulated as laboratories conducting these tests should follow some set guidelines. A strict quality control is the need of the hour. Stress must be on the necessary precautions that should be taken while conducting the various biotechniques. The analysis should be preferably repeated to ensure its validity and reliability. For getting accurate results sloppy laboratory practices need to be checked. In this light, the National Research Council, USA in 1992 recommended a series of safeguards for conducting D N A typing tests. The Federal Bureau of Investigation (FBI), USA along with the National Institute of Standards & Technology would be playing a major role in developing suitable standards for D N A
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DETECTIVE DNA
profiling. Further, designing a national D N A profiling databank in USA has been suggested. This would be very useful for sharing information by state and local law enforcement agencies. It is also suggested that laboratories performing D N A typing should be regularly tested by a government agency for complying to these standards. Setting up of these vigorous standards for forensic DNA typing is what the courts heed for guidance. The hurdles to hi-tech are surely not insurmountable. They can be overcome. However, all this should not disguise the promise of DNA profiling. Just as any scientific revolution, DNA profiling, if performed within a framework of standard guidelines would provide the multifarious field of diagnostics the most reliable, foolproof and infallible tool having a rich store of promises for the future. Lets take our hats off for the hero the detective D N A that indeed plays a stupendous and versatile role in this technology.
Glossary
Antigen: Any substance whose entry into an organism provokes the synthesis of molecules called antibodies. These antibodies are so specific that they bind only to those antigens in response to which they are formed. Archaeology: The scientific study of the material remains of man's past. Exonuclease: An enzyme which cleaves nucleotides, one at a time, from the end of a polynucleotide chain. Scintillation counter: A detector having a photosensitive region which detects and measures the energy of a particle or a radiation emitted by a radioactive substance. Specific activity: It is the activity per unit mass of a pure radioactive substance and is expressed in Curies per gram.
is the technology of DNA profiling, as it unerringly reveals the identity of any organism from just a tiny body specimen. Commonly called DNA fingerprinting, it is widely popular in parentage testing and forensic identification, as it involves the analysis of the 'blueprint' for establishing a person's unique molecular identity. Not just that. The causative bug of several infectious diseases can be now detected with utmost precision. Ill-fated genes responsible for many crippling and fatal genetic disorders can be predetermined before they spell disaster. Any alteration in the blueprint causing these disorders can be identified. Early diagnosis of a genetic disorder, even in an unborn child, is possible today. This lucidly written and profusely illustrated book, targeted at laymen explains with utmost clarity the sophisticated modern biotechniques involved in DNA profiling and its wide range of applications. This ushering in of a new era of diagnostics owes its tribute to none other but the key molecular investigator of DNA profiling, the modern day Sherlock Holmes the detective DNA. About the Author
Parvinder Chawla (born 1966) graduated with Zoology (Honours) from Gargi College, New Delhi where she topped and secured the third position in the University of Delhi. After doing her M. Biotechnology from Ail India Institute of Medical Sciences (A.I.I.M.S), New Delhi, she joined the National Institute of Science Communication, then Publications & Information Directorate (CSIR) in 1991 as a scientist. Engaged in the popularization of science for the last seven years, she has so far written about 100 popular science articles which have appeared in various newspapers and magazines. She has also edited 10 books on various topics. Detective DNA is her first popular science book.
INFALLIBLE
9 ISBN : 81-7236-175-0

Detective DNA Parwinder Chawla 1998

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DETECTIVE DNA

CSIR GOLDEN JUBILEE SERIES

Detective DNA Parvinder Chawla

...46 ...66 ...80 . . . 104 . . . 118 . . . 126 . . . 133

The Ultimate Identity

THE ULTIMATE IDENTITY

The ABO system of matching blood groups

White blood cells

The drumstick is unique to women

THE ULTIMATE IDENTITY

THE ULTIMATE IDENTITY

Skin tissue Bone marrow cells

Loop Arch Whorl

The four standard fingerprint patterns

THE ULTIMATE IDENTITY

THE ULTIMATE IDENTITY 21

W h o is the actual mother? King Solomon finds out

THE ULTIMATE IDENTITY

THE ULTIMATE IDENTITY

Broad \ Applications of DNA Profiling y \

The Power of Repeats

THE POWER OF REPEATS

THE POWER OF REPEATS Base pair

Nitrogenous base Phosphate (P) i Deoxyribose sugar (S)

T h e structure of the d o u b l e helix. C o m p l e m e n t a r y b a s e s bind to e a c h o t h e r just as p r o n g s fit into sockets (inset)

"~~Tandetnly repeated sequences

THE POWER OF REPEATS

DETECTIVE DNA Gene for antibiotic gene resistance

THE POWER OF REPEATS

Tone arm Pick up

THE POWER OF REPEATS

Length (in Kilobases)

Pattern of inheritance of a variable stretch of D N A over three generations

THE POWER OF REPEATS

Consecutively repeated segment (VNTR)

THE POWER OF REPEATS

THE POWER OF REPEATS

Gifting the Torch

GIFTING THE TORCH 45

mentary single strand of DNA

Changes in temperature can unwound and rewound DNA strands

GIFTING THE TORCH

GIFTING THE TORCH

A radioactively labelled nucleotide gets inserted in the DNA strand.

The process continues

A schematic representation of the nick translation reaction

Nick translation reaction

0.5-1.0 |ig (ljig = 10"6 gram)

Mixture of unlabelled nucleotides (say, dATP, dGTP and dTTP)

5}il (1^1 = 10"6 litre)

oc32P-labelled CTP (50|xCurie/|xl)

DNase I + DNA Polymerase I

Distilled water to make total volume:

GIFTING THE TORCH

A mixture of large and small molecules applied to a column of dextran beads

Small molecules penetrate into the pores of beads Dextran bead

GIFTING THE TORCH

GIFTING THE TORCH

The Master Plan

THE MASTER PLAN

Vacuum Refrigeration Motor

(a) Centrifugal force -

Solvent Small sized particles Medium sized particles

Large sized particles