International Journal Of Human Genetics Medical Biotechnology and Microbiological Studies

Volume 1, Issue 1, August 2012

24

DNA Fingerprinting and Allied Techniques in Forensic Studies
Priya Ramprasanth
1
Neha Solanke
2

2

Department of Biological Sciences


University of Nottinghamshire


United Kingdom


3 University of Leicester


Preston, United Kingdom

priya.dhiman555@gmail.com




Abstract Forensic analysis deals with DNA
extracted from samples in a crime scene, such as
from a dried blood stain and using it to analyse
the particular evidences for linking them to the
possible suspects. How the major problems of
DNA fragmentation and DNA degradation are
overcome form the major crux of the paper. It
covers majority of the techniques used worldwide.
Recent high profile court cases have put the
spotlight on DNA forensics and created the
impression that there is a lack of agreement
among the experts on the reliability of this
evidence. Hence this review shall try to highlight
the importance of DNA fingerprinting and allied
techniques in forensic studies.

Keywords-DNA Fingerpriting, AFLP,
RFLP, Mitochondrial Fragmentatino
I. Introduction
DNA (deoxyribonucleic acid) represents the
blueprint of the human genetic makeup. It exists in
virtually every cell of the human body and differs
in its sequence of nucleotides (molecules that
make up DNA, also abbreviated by letters, A, T,
G, C; or, adenine, thymine, guanine, and cytosine,
respectively). The human genome is made up of 3
billion nucleotides, which are 99.9% identical
from one person to the next. The 0.1% variation,
therefore, can be used to distinguish one individual
from another. It is this difference that can be used
by forensic scientists to match specimens of blood
, tissue, or hair follicles to an individual with a
high level of certainty.
The complete DNA of each individual is
unique, with the exception of identical twins. A
DNA fingerprint, therefore, is a DNA pattern that
has a unique sequence such that it can be
distinguished from the DNA patterns of other
individuals. DNA fingerprinting is also called
DNA typing.
DNA fingerprinting was first used for sample
identification after the geneticist Alec J. Jeffreys
from the University of Leicester in Great Britain
discovered that there are patterns of genetic
material that are unique to almost every individual.
He called these repetitive DNA sequences
"minisatellites." The two major uses for the
information provided by DNA-fingerprinting
analysis are for personal identification and for the
determination of paternity.
DNA fingerprinting is based on DNA analyzed
from regions in the genome that separate genes
called introns. Introns are regions within a gene
that are not part of the protein the gene encodes.
They are spliced out during processing of the
messenger RNA, which is an intermediate
molecule that allows DNA to encode protein. This
is in contrast to DNA analysis looking for disease
causing mutations, where the majority of
mutations involve regions in the genes that code
for protein called exons. DNA fingerprinting
usually involves introns because exons are much
more conserved and therefore, have less variability
in their sequence.
DNA fingerprinting was originally used to
identify genetic diseases by linking disease genes
within a family based on the inheritance of the
segregating markers and the likelihood that they
would be in close proximity, but it also became
used for criminal investigations and forensic
science. In general, the United States courts accept
the reliability of DNA analysis and have included
these results into evidence in many court cases.
However, the accuracy of the results, the cost of
Apoorva Baluapuri
3
1 Fourth Dimension ,Chennai , India
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Volume 1, Issue 1, August 2012
25

testing, and the misuse of the technique have made
it controversial.
In forensics laboratories, DNA can be analyzed
from a variety of human samples including blood,
semen , saliva , urine, hair, buccal (cheek cells),
tissues, or bones. DNA can be extracted from
these samples and analyzed in a lab and results
from these studies are compared to DNA analyzed
from known samples. DNA extracted from a
sample obtained from a crime scene then can be
compared and possibly matched with DNA
extracted from the victim or suspect.
DNA can be extracted from two different
sources within the cell. DNA found in the nucleus
of the cell, also called nuclear DNA (nDNA) is
larger and contains all the information that makes
us who we are. It is tightly wound into structures
called chromosomes. DNA can also be found in an
organelle within the cell called the mitochondria,
which functions to produce energy that drives all
the cellular processes necessary for life.
Mitochondrial DNA (mtDNA) is much smaller,
contains only 16,569 nucleotide bases (compared
with nDNA, which contains 3.9 billion) and it is
not wound up into chromosomes. Instead, it is
circular and there are many copies of it.
Nuclear DNA is analyzed in evidence
containing blood, semen, saliva, body tissues, and
hair follicles. DNA from the mitochondria,
however, is usually analyzed in evidence
containing hair fragments, bones, and teeth.
Mitochondrial DNA analysis is typically
performed in cases where there is an insufficient
amount of sample, the nDNA is uninformative, or
if supplemental information is necessary.
Unlike nDNA, where one copy of a
chromosome comes from the father and the other
from the mother, mtDNA is exclusively inherited
from the maternal side. Therefore, the maternal
mtDNA should be the same as her offspring. This
can be helpful in cases where it is not possible to
obtain a sample from the suspect but it is possible
to obtain a sample from one of the suspect's
biologically related family members. By doing so,
the suspect can be excluded as the culprit of a
crime if the results indicate that the relevant family
member's mtDNA does not match the mtDNA
fingerprint from the sample.
Mitochondrial DNA can be informative in a
different way than nDNA. Less than 10% of the
mitochondrial genome is noncoding and localized
in a region called the D-loop. In this region, there
are sequence variations that are inherited that can
be used for forensic purposes. These regions,
called hypervariable regions, are broken down into
two sections: HV1 and HV2. It is within these
regions that inherited sequence variations can be
identified.
One of the main reasons mtDNA analysis can
be helpful to forensic scientists is that in some
tissues, mitochondrial DNA is in excess compared
to nDNA. As nDNA exists in chromosomes and
there are only two copies of each chromosome
(one inherited maternally, the other paternally) per
cell, the nDNA copy number is much smaller. The
mitochondrial genome can have a copy number of
210 per organelle and in some cases the number
of organelles can reach the hundreds. For example,
in muscle tissue, where the demand for energy is
highest, there are a larger number of copies of the
mitochondrial genome. Analysis of mtDNA,
therefore, can be particularly helpful in forensic
cases where sample integrity or size is
compromised or when confirmation is needed.
There are many methods that forensic scientists
use to determine the sample's DNA fingerprint.
Once DNA is extracted, it can then be analyzed
using a variety of molecular genetics techniques.
In some cases, there is not enough DNA to directly
evaluate it. If this occurs, a technique called the
polymerase chain reaction (PCR ) is used to
amplify the genomic DNA from a sample. This
procedure allows a scientist to amplify a specific
sequence of DNA in the genome exponentially, so
that it is in large enough quantities to be analyzed.
DNA analysis can be performed by sequencing
the amplified DNA fragment using fluorescently
labeled nucleotides and a laser that will recognize
the nucleotide based on the fluorescent label to
which it is attached. This technique is expensive,
may not be informative, and is generally not the
best approach to DNA fingerprint a sample.
If there is enough DNA, the DNA extracted
from the sample can be cut or segmented using
specific enzymes (proteins that speed up chemical
reactions) called restriction endonucleases that act
as molecular scissors by cutting specific sequences
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Volume 1, Issue 1, August 2012
26

that they recognize. By cutting in the same
sequence that is present in different locations
throughout the genome, a pattern of fragments can
be formed. Differences in the sequence patterns
between two samples can be due to inherited
variations in the DNA that can distinguish two
different samples.
Once the DNA is cut, the segments are
arranged by size using a process called
electrophoresis, whereby an electrical field is
generated, pulling the negatively charged DNA
toward the positively charged end through a gel-
like matrix. The segments are marked with
radioactive probes and exposed on x-ray film,
where they form a characteristic pattern of black
bars. This pattern is called the DNA fingerprint. If
the DNA fingerprints produced from two different
samples match, the two samples are likely to have
come from the same person.
DNA can also be processed and cut with
restriction enzymes. If there is a variation in a
particular sequence that results in the enzyme no
longer recognizing and cutting the DNA (or a loss
of the cut site), a larger fragment will be observed
when running the DNA in a gel by electrophoresis.
Using a chemical that binds to DNA (called
ethidium bromide) and fluoresces when it is
excited by ultraviolet radiation, the fragments can
be observed on a gel based on size. Bigger
fragments will migrate more slowly in the gel. An
individual with the sequence variation in which
the enzyme does not cut would have a longer size
fragment than the individual with the variation the
enzyme does cut.
The original DNA fingerprinting procedure
used Variable Number Tandem Repeats (VNTR),
which are repetitive DNA sequences that are
spread throughout the genome in noncoding
regions. These targets are large, with repeat
numbers that are variable from person to person
and have a repeat size composed of hundreds of
nucleotides which can be repeated a hundred
times.
The biggest problem with using the VNTR-
fingerprinting approach is that DNA extracted
from samples in a crime scene, such as from a
dried blood stain, is often broken up into tiny
pieces due in most cases to natural DNA-
degrading processes. This can make DNA analysis
difficult, unless informative fragments remain
intact. Additionally, the smaller the sample, the
more likely it will be degraded. For example, a
plucked hair might contain up to 30 nanograms
(30 ng, or 30 billionths of one gram) of genomic
DNA, but a hair shaft without the root might
maximally only contain 0.1 ng of DNA. The
integrity of the sample as well as the quantity,
therefore, can make reliable and definitive identity
determination difficult.
More recent approaches have circumvented the
problem associated with degraded DNA. Shorter
repetitive sequences, or short tandem repeats
(STR), were later identified and found to contain
repeat core units of three, four, or five nucleotides
long and have a complete length of only 80400
nucleotides. Due to the shortness of these
sequences, only 50 pg of DNA (which is almost a
1000 times less than that found in a hair shaft
without the root) is required. The discriminating
power, when analyzing STRs at multiple locations
with the genome, can match persons with a
probability of 1 in 1015 to a stain. The DNA
fingerprint using STR analysis can, therefore, be
an extremely powerful technique in forensic
sciences.
With the completion of the human genome
sequence and the rapid post-genomic
characterization of the sequences, it has become
easier to analyze samples pertinent for forensic
applications. In fact, forensic scientists have been
able to link a suspect to the scene of a crime using
dried chewing gum, the cells in the saliva from the
butt of a cigarette, and cells found underneath
fingernails. DNA fingerprinting, therefore, has
revolutionized the forensic sciences by its use in
investigations and prosecutions of active criminal
cases, missing persons investigations, re-
examining dead-end cases, post-conviction
exoneration, and studies where maternal
relatedness is in question.
II. RFLP analysis
The first methods for finding out genetics used
for DNA profiling involved restriction enzyme
digestion, followed by Southern blot analysis.
Although polymorphisms can exist in the
restriction enzyme cleavage sites, more commonly
the enzymes and DNA probes were used to
analyze VNTR loci. However, the Southern blot
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technique is laborious, and requires large amounts
of undegraded sample DNA. Also, Karl Brown's
original technique looked at many minisatellite
loci at the same time, increasing the observed
variability, but making it hard to discern individual
alleles (and thereby precluding parental testing).
These early techniques have been supplanted by
PCR-based assays.
III. PCR ANALYSIS
With the invention of the polymerase chain
reaction (PCR) technique, DNA profiling took
huge strides forward in both discriminating power
and the ability to recover information from very
small (or degraded) starting samples. PCR greatly
amplifies the amounts of a specific region of
DNA, using oligonucleotide primers and a
thermostable DNA polymerase. Early assays such
as the HLA-DQ alpha reverse dot blot strips grew
to be very popular due to their ease of use, and the
speed with which a result could be obtained.
However they were not as discriminating as RFLP.
It was also difficult to determine a DNA profile for
mixed samples, such as a vaginal swab from a
sexual assault victim.
Fortunately, the PCR method is readily
adaptable for analyzing VNTR loci. In the United
States the FBI has standardized a set of 13 VNTR
assays for DNA typing, and has organized the
CODIS database for forensic identification in
criminal cases. Similar assays and databases have
been set up in other countries. Also, commercial
kits are available that analyze single nucleotide
polymorphisms (SNPs). These kits use PCR to
amplify specific regions with known variations
and hybridize them to probes anchored on cards,
which results in a colored spot corresponding to
the particular sequence variation.
IV. STR analysis
The method of DNA profiling used today is
based on PCR and uses short tandem repeats
(STR). This method uses highly polymorphic
regions that have short repeated sequences of
DNA (the most common is 4 bases repeated, but
there are other lengths in use, including 3 and 5
bases). Because different unrelated people have
different numbers of repeat units, these regions of
DNA can be used to discriminate between
unrelated individuals. These STR loci (locations)
are targeted with sequence-specific primers and
are amplified using PCR. The DNA fragments that
result are then separated and detected using
electrophoresis. There are two common methods
of separation and detection, capillary
electrophoresis (CE) and gel electrophoresis.
The polymorphisms displayed at each STR
region are by themselves very common, typically
each polymorphism will be shared by around 5 -
20% of individuals. When looking at multiple loci,
it is the unique combination of these
polymorphisms to an individual that makes this
method discriminating as an identification tool.
The more STR regions that are tested in an
individual the more discriminating the test
becomes.
From country to country, different STR-based
DNA-profiling systems are in use. In North
America systems which amplify the CODIS 13
core loci are almost universal, while in the UK the
SGM+ system, which is compatible with The
National DNA Database in use. Whichever system
is used, many of the STR regions under test are the
same. These DNA-profiling systems are based
around multiplex reactions, whereby many STR
regions will be under test at the same time.
Capillary electrophoresis works by
electrokinetically (movement through the
application of an electric field) injecting the DNA
fragments into a thin glass tube (the capillary)
filled with polymer. The DNA is pulled through
the tube by the application of an electric field,
separating the fragments such that the smaller
fragments travel faster through the capillary. The
fragments are then detected using fluorescent dyes
that were attached to the primers used in PCR.
This allows multiple fragments to be amplified
and run simultaneously, a procedure known as
multiplexing. Sizes are assigned using labeled
DNA size standards that are added to each sample,
and the number of repeats are determined by
comparing the size to an allelic ladder, a sample
that contains all of the common possible repeat
sizes. Although this method is expensive, larger
capacity machines with higher throughput are
being used to lower the cost/sample and reduce
backlogs that exist in many government crime
facilities.
Gel electrophoresis acts using similar principles
as CE, but instead of using a capillary, a large
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polyacrylamide gel is used to separate the DNA
fragments. An electric field is applied, as in CE,
but instead of running all of the samples by a
detector, the smallest fragments are run close to
the bottom of the gel and the entire gel is scanned
into a computer. This produces an image showing
all of the bands corresponding to different repeat
sizes and the allelic ladder. This approach does not
require the use of size standards, since the allelic
ladder is run alongside the samples and serves this
purpose. Visualization can either be through the
use of fluorescently tagged dyes in the primers or
by silver staining the gel prior to scanning.
Although it is cost-effective and can be rather high
throughput, silver staining kits for STRs are being
discontinued. In addition, many labs are phasing
out gels in favor of CE as the cost of machines
becomes more manageable.
The true power of STR analysis is in its
statistical power of discrimination. In the US, there
are 13 core loci (DNA locations) that are currently
used for discrimination in CODIS. Because these
loci are independently assorted (having a certain
number of repeats at one locus doesn't change the
likelihood of having any number of repeats at any
other locus), the product rule for probabilities can
be applied. This means that if someone has the
DNA type of ABC, where the three loci were
independent, we can say that the probability of
having that DNA type is the probability of having
type A times the probability of having type B
times the probability of having type C. This has
resulted in the ability to generate match
probabilities of 1 in a quintillion (1 with 18 zeros
after it) or more. However, since there are about
12 million monozygotic twins on Earth, that
theoretical probablitity is useless. For example, the
actual probability that 2 random persons have the
same DNA is only 1 in 3 trillion

V. Mitochondrial Analysis
For highly degraded samples, it is sometimes
impossible to get a complete profile of the 13
CODIS STRs. In these situations, mitochondrial
DNA (mtDNA) is sometimes typed due to there
being many copies of mtDNA in a cell, while there
may only be 1-2 copies of the nuclear DNA.
Forensic scientists amplify the HV1 and HV2
regions of the mtDNA, then sequence each region
and compare single nucleotide differences to a
reference. Because mtDNA is maternally
inherited, directly linked maternal relatives can be
used as match references, such as one's maternal
grandmother's sister's son. A difference of two or
more nucleotides is generally considered to be an
exclusion. Heteroplasmy and poly-C differences
may throw off straight sequence comparisons, so
some expertise on the part of the analyst is
required. mtDNA is useful in determining unclear
identities, such as those of missing persons when a
maternally linked relative can be found. mtDNA
testing was used in determining that Anna
Anderson was not the Russian princess she had
claimed to be, Anastasia Romanov.
mtDNA can be obtained from such material as
hair shafts and old bones/teeth
VI. AmpFLP
Another technique, AmpFLP, or amplified
fragment length polymorphism was also put into
practice during the early 1990s. This technique
was also faster than RFLP analysis and used PCR
to amplify DNA samples. It relied on variable
number tandem repeat (VNTR) polymorphisms to
distinguish various alleles, which were separated
on a polyacrylamide gel using an allelic ladder (as
opposed to a molecular weight ladder). Bands
could be visualized by silver staining the gel. One
popular locus for fingerprinting was the D1S80
locus. As with all PCR based methods, highly
degraded DNA or very small amounts of DNA
may cause allelic dropout (causing a mistake in
thinking a heterozygote is a homozygote) or other
stochastic effects. In addition, because the analysis
is done on a gel, very high number repeats may
bunch together at the top of the gel, making it
difficult to resolve. AmpFLP analysis can be
highly automated, and allows for easy creation of
phylogenetic trees based on comparing individual
samples of DNA. Due to its relatively low cost and
ease of set-up and operation, AmpFLP remains
popular in lower income countries.

VII. Y-chromosome analysis
Recent innovations have included the creation
of primers targeting polymorphic regions on the
Y-chromosome (Y-STR), which allows resolution
of a mixed DNA sample from a male and female
and/or cases in which a differential extraction is
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not possible. Y-chromosomes are paternally
inherited, so Y-STR analysis can help in the
identification of paternally related males. Y-STR
analysis was performed in the Sally Hemings
controversy to determine if Thomas Jefferson had
sired a son with one of his slaves.
VIII. DNA databases
There are now several DNA databases in
existence around the world. Some are private, but
most of the largest databases are government
controlled. The United States maintains the largest
DNA database, with the Combined DNA Index
System, holding over 5 million records as of
2007.The United Kingdom maintains the National
DNA Database (NDNAD), which is of similar
size. The size of this database, and its rate of
growth, is giving concern to civil liberties groups
in the UK, where police have wide-ranging powers
to take samples and retain them even in the event
of acquittal.
The U.S. Patriot Act of the United States
provides a means for the U.S. government to get
DNA samples from other countries if they are
either a division of, or head office of, a company
operating in the U.S. Under the act, the American
offices of the company can't divulge to their
subsidiaries/offices in other countries the reasons
that these DNA samples are sought or by whom.
When a match is made from a National DNA
Databank to link a crime scene to an offender who
has provided a DNA Sample to a databank that
link is often referred to as a cold hit. A cold hit is
of value in referring the police agency to a specific
suspect but is of less evidential value than a DNA
match made from outside the DNA Databank.

IX. Considerations when evaluating DNA
evidence
In the early days of the use of genetic
fingerprinting as criminal evidence, juries were
often swayed by spurious statistical arguments by
defense lawyers along these lines: given a match
that had a 1 in 5 million probability of occurring
by chance, the lawyer would argue that this meant
that in a country of say 60 million people there
were 12 people who would also match the profile.
This was then translated to a 1 in 12 chance of the
suspect being the guilty one. This argument is not
sound unless the suspect was drawn at random
from the population of the country. In fact, a jury
should consider how likely it is that an individual
matching the genetic profile would also have been
a suspect in the case for other reasons. Another
spurious statistical argument is based on the false
assumption that a 1 in 5 million probability of a
match automatically translates into a 1 in 5 million
probability of innocence and is known as the
prosecutor's fallacy.
When using RFLP, the theoretical risk of a
coincidental match is 1 in 100 billion
(100,000,000,000), although the practical risk is
actually 1 in 1000 because monozygotic twins are
0.2% of the human population. Moreover, the rate
of laboratory error is almost certainly higher than
this, and often actual laboratory procedures do not
reflect the theory under which the coincidence
probabilities were computed. For example, the
coincidence probabilities may be calculated based
on the probabilities that markers in two samples
have bands in precisely the same location, but a
laboratory worker may conclude that similarbut
not precisely identicalband patterns result from
identical genetic samples with some imperfection
in the agarose gel. However, in this case, the
laboratory worker increases the coincidence risk
by expanding the criteria for declaring a match.
Recent studies have quoted relatively high error
rates which may be cause for concern. In the early
days of genetic fingerprinting, the necessary
population data to accurately compute a match
probability was sometimes unavailable. Between
1992 and 1996, arbitrary low ceilings were
controversially put on match probabilities used in
RFLP analysis rather than the higher theoretically
computed ones. Today, RFLP has become widely
disused due to the advent of more discriminating,
sensitive and easier technologies.
STRs do not suffer from such subjectivity and
provide similar power of discrimination (1 in
10^13 for unrelated individuals if using a full
SGM+ profile) It should be noted that figures of
this magnitude are not considered to be
statistically supportable by scientists in the UK,
for unrelated individuals with full matching DNA
profiles a match probability of 1 in a billion (one
thousand million) is considered statistically
supportable (Since 1998 the DNA profiling system
supported by The National DNA Database in the
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30

UK is the SGM+ DNA profiling system which
includes 10 STR regions and a sex indicating test.
However, with any DNA technique, the cautious
juror should not convict on genetic fingerprint
evidence alone if other factors raise doubt.
Contamination with other evidence (secondary
transfer) is a key source of incorrect DNA profiles
and raising doubts as to whether a sample has been
adulterated is a favorite defense technique. More
rarely, Chimerism is one such instance where the
lack of a genetic match may unfairly exclude a
suspect. .
X. Evidence of genetic relationship
It's also possible to use DNA profiling as
evidence of genetic relationship, but testing that
shows no relationship isn't absolutely certain.
While almost all individuals have a single and
distinct set of genes, rare individuals, known as
"chimeras", have at least two different sets of
genes. There have been several cases of DNA
profiling that falsely "proved" that a mother was
unrelated to her children.[8]
In case of fake DNA evidence, the value of
DNA evidence has to be seen in light of recent
cases where criminals planted fake DNA samples
at crime scenes. In one case, a criminal even
planted fake DNA evidence in his own body: Dr.
John Schneeberger raped one of his sedated
patients in 1992 and left semen on her underwear.
Police drew Schneeberger's blood and compared
its DNA against the crime scene semen DNA on
three occasions, never showing a match. It turned
out that he had surgically inserted a Penrose drain
into his arm and filled it with foreign blood and
anticoagulants.paper.
XI. Familial searching
Familial searching is the use of family
members' DNA to identify a closely related
suspect in jurisdictions where large DNA
databases exist, but no exact match has been
found. The first successful use of the practice was
in a UK case where a man was convicted of
manslaughter when he threw a brick stained with
his own blood into a moving car. Police could not
get an exact match to the UK's DNA database
because the man had no criminal convictions, but
police implicated him using a close relative's
DNA. The term DNA fingerprinting was coined
by British geneticist Alec Jeffreys only ten years
ago. Since that time, DNA forensics has become
an important tool in law enforcement. In some
cases the DNA tests have helped convict suspects,
while in others the tests have exonerated suspects
or overturned previous convictions. Recent high
profile court cases have put the spotlight on DNA
forensics and created the impression that there is a
lack of agreement among the experts on the
reliability of this evidence.

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