MOLECULAR DIAGONISTICS:

Success of modern agriculture and medicine often depends upon

on the ability of the workers in these fields to detect the presence
of specific:

Bacteria
Fungi
Parasites
Proteins and
Small molecules in humans, animal, plants, water and soil
For example:

The prevention, control and treatment of infectious

diseases is generally facilitated by

Early and Accurate identification of causative

pathogenic organisms
Many of these detection procedures require the
growth in culture of the potential pathogen and then

 Analysis of a spectrum of physical properties that facilitates its

identification
Although tests of this type are effective and reasonably
specific but they often slow and costly
These constraints apply to the identification of both bacteria
and parasitic organisms
Table 9.1 Glick, 3rd Ed

In addition, if the pathogenic organism does not grow well or

can not be cultivated
The opportunity to detect the disease-causing organism is
severely limited
For example:

Chlamydia trachomatis - an obligately intracellular

parasite, causes a sexually transmitted disease,
prevalent in North America and Europe

Clinical Diagnosis of Chlamydia trachomatis infection

is difficult because
o

long term culture is required

Frequently false negative results are obtained

and as a result adequate treatment procedures
are not implemented

Certainly if growth were required for detection, then

at best only few of all known pathogenic organisms
could ever be routinely identified

To overcome this major constrain, molecular diagnostic

procedures using either:

Immunological
DNA detection methodologies have been devised

In general, any useful detection strategy must be specific,

sensitive and simple:

Specificity means that the assay must yield a positive response

for only the target organism/molecule

Sensitivity means that the diagnostic test must identify very

small amounts of the target organism/molecule even in the
presence of other potentially interfering organisms/substances

Simplicity is required for the test to be run efficiently,

effectively and inexpensive on a routine basis

It is estimated that Worldwide sales of immuno-diagnostics

accounted for approximate US $ 7.7 billion in 1999

This figure is expected to continue to increase by 5-10% for

the next 5-10 years

The market for DNA based diagnostic procedures was around

US $ 500 million in 1999 and is

Increasing at around 30% per year so that by 2004 it is

expected to be worth approximately $ 2 billion

Now it is 2014, one can imagine the potentials

Immunological Diagnostic Procedures:

Sensitive, simple and specific

Used for wide range of applications. i.e.

Drug testing

Assessment and monitoring of various

cancers

Detection of specific metabolites and

Pathogenic identification and monitoring

However there are some limitations like:

If target is protein, then the use of antibodies

requires that the genes contributing to the presence of
the target site be expressed and

that the target site not be blocked or masked in

any way that would prevent the binding of the
antibody

Bases of immunological diagnostic procedures are

.?

There are number of different ways to determine

whether an antibody has bound to its target antigen?
One of the methods is
Enzyme Linked Immunosorbent Assay (ELISA):

1st antibody/ primary antibody rabbit that has

immunized with target antigen
Secondary Antibody goat immunized with rabbit
antibodies conjugated with alkaline phosphate,
peroxidase or urease
Which converts colorless substrate to colored
products

Fig 9.1 Glick 3rd Ed

Polyclonal Antibodies:

Amount of different antibodies may very

from
batch to batch
Not very specific - can not distinguished
between pathogenic and non pathogenic if
different is a single determinant

Monoclonal Antibodies:

Fig 9.2 Glick 3rd Ed /4th Ed

Fig 9.3 Glick 3rd Ed /4th Ed
Fig 9.4 Glick 3rd Ed / 4th Ed
Fig 9.5 Glick 3rd Ed / Table 9.2 4th Ed

HGPRT- myeloma and the myeloma-myeloma fusion cell can not use hypoxanthine as a precursor for the
biosynthesis of purines (guanine and adenine) which are essential for nucleic acid synthesis. But they have second
naturally occurring pathway for purine biosynthesis that utilize the enzyme dihydrofolate reductase. Therefore,
aminopterin, an inhibitor of enzyme, is included in the medium. So they die.
Spleen-myeloma fusion cells survive in HAT medium because spleen cell contributes functional HGPRT, which can
utilize the exogenous hypoxanthine even DHFR is blocked by aminopterin and because the cell division functions of
myeloma cell are active. Thymidine is provided to overcome the block in pyrimidine production that is caused by
the inhibition of DHFR by aminopterin

-In mammals, nucleotides are

produced via two alternative routes
- If the de novo pathway is blocked,
nucleotide synthesis becomes

(basic precursors i.e. sugars, amino acids)

DNA Diagnostic Systems:

What is the basis of nucleic acid hybridization?

Fig 9.6 Glick 3rd Ed / Fig 9.10 4th Ed

Rapid and reliable

Extremely specific and highly sensitive
Three critical elements are required i.e.

Probes

Target DNA

Signal Detection
Researchers successfully used probes hybridize to
target DNA from Fecal samples, Urine, Blood, Throat
washing and tissue samples without extensive DNA
purification

If a target sequence is rare in the working sample

.?
The PCR can be used to amplify it

Diagnosis of Malaria:

Plasmodium falciparum - a parasite causes malaria

affects about 1/3 of the worlds population

Parasite infects and destroys RBCs leading to the

fever and in severe cases damage to brain, kidneys and
other organs

Sensitive, simple and in expensive methods are

required to identify the sources of parasite in
various localities:

To asses the progress of eradication programs

To facilitate early treatment

Current detection procedures are:

Microscopic examination of blood smear Effective but labor intensive and time consuming

ELISA -rapid but do not discriminate between

present and past infections because

Designed simply to detect antiplasmodium antibodies

in infected individuals blood

DNA diagnostic procedure that selectively measures

only current infection i.e. the presence of DNA
containing organisms, was developed by using highly
repeated DNA from P. falciparum

 How this method has been developed..?

Genomic library of parasite DNA

Specific probes hybridized with P. falciparum but not

with P. vivax, P. cynomolgi / human DNA
This probe detected 10 pg of purified P. falciparum DNA
OR 1ng P. falciparum in blood

More than 100 different DNA diagnostic probes have been

isolated and characterized for the detection of various
Pathogenic strains of bacteria
Viruses and
Parasites
For example probes for human bacterial infections caused by:

Legionella pneumophila respiratory failure

Salmonella typhi food poisoning
Campylobacter hyointestinalis gastritis
Enterotoxigenic E.coli gastroenteritis

Detection of Trypanosoma cruzi:

The protozoan parasite is the causative agent of Chagas disease
In this disease parasite invades the liver, spleen, lymph nodes
and central nervous system, where they multiply and destroy
the parasitized cells
Common parasite in Latin America
Spread by insects and responsible for 50,000 deaths/year

 Common methods are:

Microscopic examination of fresh blood samples

Alternatively a test that takes a longer time but ensure

that the parasitic has not been overlooked entails
feeding a patients blood to uninfected insects and then
examining with a microscope the contents of the
insects intestines for parasites about 30-40 days later

Both these tests are laborious, time consuming and

costly

Immunological tests can also be used to diagnose

give false positive results

As a possible alternative to these less satisfactory

procedures, several PCR based assays have been
developed

At present, PCR assays for Chagas disease are used as

adjunct to the traditional diagnostic procedure that are
currently in widespread use

In one of the PCR-based assay procedure, 188bp DNA

fragment that is present in multiple copies in T. cruzi
genome but absent from the genomic DNA of several
related parasites is the target sequence

The presence of the amplified 188bp DNA fragment is

readily detected by polyacrylamide gel electrophoresis

In general, with minor variations in the methodology,

such as the primer sequences, PCR can facilitate
detection of a wide range of bacteria, viruses and
parasites

Currently, there are several PCR-based diagnostic kits

that have been approved for use by the US Food and
Drug Administration for detection and quantification of
HIV virus, M. tuberculosis and C. trachomatis

Nonradioactive Hybridization Procedures

Radioactive probe - 32P give high specific activity

Short lived
Potentially dangerous
Require special laboratory equipment for handling and safe
disposal
Nonradioactive probe-signal amplification by enzymatic
conversion of either chromogenic / chemiluminescent
substrates

 Chromogenic substrates - change color and

Chemiluminescent substrates give off light when they
are converted into specific product by an appropriate
enzyme
Fig. 9.7 Glick 3rd Ed / Fig 9.11 4th Ed
Advantages:
Biotin - labeled DNA: stable for at least one years at
room temperature
Devices detecting chemiluminesence sensitive

Detection of the emitted light with X-ray/ luminometer

scoring of color change within few hours

Bacterial analog of avidin a chicken egg white

protein

/peroxidase

 PCR - Based Assays:

Amplification product can be labeled by fluorescent dye like
fluoresein green and rhodamine - red under certain
wavelength of light

Fig. 9.8 Glick 3rd Ed / Fig 9.11 4th Ed

Fig. 9.9 Glick 3rd Ed / 913 4th Ed
Fig. 9.10 Glick 3rd Ed /9.14 4th Ed
Fig. 9.11 Glick 3rd Ed / 9.16 4th Ed

 Novel non-radioactive methodspecific sequence of nucleic acid

involve using molecular bacon
probes

Non-complementary to each other

25 nucleotide long
15 nucleotide complementary to
target DNA and perfect matching

complementary to each other

5 nucleotide complementary to
each other

3
Non-fluorescent

Blue

Pink/orange

Green

Red

Fluorescien

Texas red

 DNA Fingerprinting:

Used to characterize
proceedings

To establish both that;

Some suspects committed a crime and that

Some individuals had been wrongly convicted

of crime that they did not commit

Helps in determining the paternity and identify

victims of disasters

Biological samples like blood, semen, skin / hairs

Portion analyzed to ensure sufficient amount of intact

DNA (un-degraded) for intended analysis
Alternatively, with minute samples or with degraded
DNA, a set of PCR primers can be used to determine
whether two samples have the same amplified DNA bands

biological

samples

for

legal

DNA digested with a restriction enzyme, separated and

transblotted

Nitro-cellulose (NC) membranes sequentially with 4/5

separate radiolabel probes, recognize a distinct DNA
sequence by completely stripping off each probe
Fig. 9.12 Glick 3rd Ed Fig 9.17 4th Ed

Common probe used for this type of test consists of

human mini- satellite DNA

These sequences occurs throughout the human genome

and it consists of tandemly repeated sequences
Fig. 9.13 Glick 3rd Ed / Fig 9.16 4th Ed

Length of the sequence ranges from 9-40 bp and

number of repeats in the mini-satellites ranges from
10-30

Since each hybridization and

autoradiography step can take up to
10-14 days, the entire process may
take many weeks and even several
months

The mini-satellite DNA sequences at a specific

chromosome location can have different lengths is
different individuals

This variability in length of mini-satellite DNA in

different individuals is due to either gain or loss of
tandem repeats during DNA replication

These changes do not have any biological affect as it

does not code any protein

Unrelated individuals generally have mini-satellite that

differs in length but

Children will inherit one set of mini-satellite sequence

from one parent and other from other parent

A mini satellite DNA pattern (fingerprint) represents

repertoire of the lengths of some of these sequences in
an individual

Because of extensive variability in human mini-satellite

sequences, the chance of finding two individuals in the
population with same DNA fingerprint is about 1 into
105 -108

Therefore, an individuals DNA banding pattern based

on mini-satellite DNA sequence is almost as unique as
his or her finger print

In addition to forensic application of DNA fingerprinting, this technique is also used to determine
paternity issues of a child

Amount of DNA in forensic sample is quite small but

the DNA is relatively degraded - PCR

 Random Amplified Polymorphic DNA (RAPD)

DNA banding pattern is not only important for forensic

analyses but

Also useful in distinguishing among different plant

cultivars
Achieved by using random amplified polymorphic
DNA makers, in this procedure:

Arbitrary oligonucleotide primers

9-10 bp long
Do not contain palindromic sequence
G + C content of 50% - 80%
are individually added to a sample of plant
chromosomal DNA

Each PCR is initiated by addition of a single primer, so

the same nucleotides must be able to bind to opposite
strands on the target DNA

Whenever a primer can hybridize to both strands of the

target DNA in proper orientation and two sites are
about 100 - 3000 bp from each other, then the intervening DNA will be amplified and characteristically
sized DNA fragment will be synthesize which can be
visualized following polyacrylamide gel electrophoresis

The number of amplified DNA fragments in a sample is

dependent on the primer and the genomic DNA used

Each time the same primer is used with the same target
DNA, the amplified product will be the same

A single nucleotide substitution in a primer will result in

a complete change in RAPD pattern

Thus, RAPD finger prints of different plant cultivars can

be compared to when the same set of oligonucleotide
primers is used

Fig 19.4 Glick 3rd Ed

To fingerprint the DNA of two very similar plant

strains or cultivars, it is often necessary to perform the
RAPD procedure with a number of different arbitrary
primers of known sequence until differences are
revealed
Fig. 9.15 Glick 3rd Ed

Like other molecular markers, RAPDs can be used to

characterize whole genomes, individual chromosomes
or less commonly, specific genes

ADVANTAGES:

Same (universal) set of oligonucleotide primers

can be used for all plant species

No genomic libraries, radioactivity, Southern

blotting or DNA hybridization reactions are
required

So large number of samples may be easily and

rapidly characterized and

The process can be automated

Moreover, with conventional PCR analysis, it is

necessary to know the sequence of specific
genes or gene segment that is target for
amplification

On the other hand, amplification in RAPD

analysis occurs anywhere in genome that
contains two sequences complementary to the
primer and within length limits of PCR

With this technology scientists were able to

distinguish six inbred maize lines from each
other and

maize hybrids produced by genetic crossings of

these inbred lines were shown to have the PCR
products of their parental inbred lines

RAPD markers have also been used to screen

different strains of the fungus Leptosphaeria
maculans which is the causal agent blackleg
disease in crucifers

Differences between avirulent (non-disease

causing) and virulent (disease-causing) strains
could be distinguished on the basis of specific
RAPD markers, making it easier for scientist to
produce an avirluent strain that could be used as
a biological control agent that helps to prevent
blackleg disease

 Bacterial Biosensors:

There is a need for methods that can easily and rapidly

detect the large numbers of potentially toxic
compounds that contaminate the environment

Once the contaminated sites have been identified and

their range has been delineated, then their are a number
of highly sophisticated analytical techniques available
to identify and quantify specific pollutants

Bacteria that are constitutively bioluminescent are good

candidates for pollutant directors

In the presence of pollutants, the bioluminescent

decreases, providing a clear indication of the presence
of pollutants

Naturally bioluminescent bacteria, such as marine

bacterium, Vibro fischeri, require saline conditions and
a particular pH range and are therefore not useful for
testing terrestrial ground water

However, structural genes encoding enzymes that lead

to bioluminescence (luxCDABE) may be inserted in to
random sites in the chromosomal DNA of a soil
bacterium such as Pseudomonas fluorescence

These genes do not contain a transcriptional promoter,

so after insertion in chromosomal DNA of P.
fluorescence the only luminescent colonies (visualized
in darkroom) are those in which the lux genes are
inserted downstream from a constitutive P. fluorescence
promoter

The cells that luminescence to the greatest extent and

have grown rate similar to that of the wild-type strain
are selected for testing with various environmental
pollutants; both metals and organic compounds

A suspension of bioluminescent P. fluorescence is

mixed with the solution being tested and after a 15
minute incubation together, the luminescence of the
suspension is measured in a luminometer
Fig. 9.16 Glick 3rd Ed / 4th Ed

When a test sample contains a low to moderate level of

certain pollutants, the cell luminescence is inhibited

Since this procedure is rapid, simple and inexpensive it

is a good first screen for assessing the presence of
pollution at a particular site

After a positive response with a bacterial biosensor, the

actual pollutants can be determined by other methods

Molecular Diagnosis of Genetic Disease:

DNA analysis can be used for the:

Identification of carriers of hereditary disorders
Parental diagnosis of serious genetic conditions
Early diagnosis before the onset of symptoms

Test at DNA level are definitive for determining the

existence of specific genetic mutations

Earlier, genetic testing relied almost exclusively on

biochemical assays that scored either the presence/
absence of a genetic product

DNA-based does not, however, require expression for

the detection of the mutant gene, thereby making it

theoretically possible to develop screening assays for

all single-gene diseases

Sickle - Cell Anemia:

It is a genetic disease that is the result of a single

nucleotide change (Glu
Val) in the codon for the 6th
amino acid of -chain of hemoglobin molecule
Fig 5.13 Zubay

In individuals homozygous for the defect (S/S), the

shape of red blood cells is irregular i.e. sickle shaped

Because the conformation of the hemoglobin molecule

is distorted by a single amino acid change
Fig 2.3 Peters

Biological ramifications of this genetic alterations are:

Severe anemia and

Progressive damage to the heart, lungs, brains, joints

and major organ systems
Anemia is caused by the inability of the mutated
hemoglobin to carry sufficient oxygen as a result

Life expectancy for S/S homozygotes is quite short

Heterozygous individuals (A/S i.e. genetic carriers)

have normal-shaped red blood cells and no symptoms
unless subjected to extreme conditions such as:

High altitude or
Extremes of temperature that lowers the oxygen
supply

 If both parents are heterozygous (A/S), there is a 25%

chance a that child of theirs will have sick cell anemia i.e.
will be an S/S homozygote
The sickle cell anemia gene occurs with a high frequency
among black Africans and their decedents and in Hispanic
populations
Carrier screening for the sickle-cell anemia gene is
routinely conducted in USA
so that those individual who are at risk for transmitting
this gene to their offspring can be identified

The single nucleotide change in the -globin gene that causes

sickle-cell anemia by chance abolishes a Cvn1 restriction
endonuclease site
This enzyme recognizes the sequence CCTNAGG and cleave
the DNA between C and the T where N could be any one

 In the normal gene, the DNA sequence is CCTGAGG

while in the sickle-cell anemia gene the sequence is
CCTGTGG
This difference forms the basis for a DNA diagnostic
assay
Fig 9.17 Glick 3rd Ed
With the help of this procedure, the genetic make up of
tested person can be determined

Quickly
Directly and
Easily
Because of the fortuitous loss of the Cvn1 site, this assay
functions without the need for a target probe hybridization
reaction

PCR/OLA Procedures:
Obviously, not all genetic changes that produce defective
genes affect existing restriction endonuclease sites
Therefore, other strategies for detecting single nucleotide
changes are required
One of these procedures combines PCR with an oligonucleotide ligation assay (OLA) called PCR/OLA

In normal gene at a specific site for example nucleotide

106 the pairing is A.T
In mutant gene at a specific site for example nucleotide
106 the pairing is G.C
Fig 9.18 Glick 3rd Ed

With two pairs of probes, it is possible to ascertain the

genetic make up of any tested individual

For example:

Heterozygous individuals yield positive results

with both pairs of probes

The DNA from people with two copies of the

normal gene gives a positive response only with
the set of probes that contains the nucleotide
complementary to the nucleotide at normal site

Finally, DNA from individuals with two altered

gene copies will give a positive response only
with the set of probes that is designed to detect
the mutant site

To minimize the amount of the original sample DNA

that is required for the assay, the segment of the target
DNA sequence that contains the nucleotide site to be
tested is amplified by PCR before the hybridization

The PCR/OLA system is designed to distinguish

between two possibilities i.e. ligation/no ligation and
overall it is
Rapid
Sensitive and
Highly specific

Further it has been automated with a robotic workstation to carry out the steps of the assay procedures

Under these conditions, as many as 1,200 ligation

reactions can be conducted per day

Padlock Probes:

A padlock probe is an oligonucleotide that is

complementary to a target (DNA or RNA) sequence
at its 5 and 3 ends but not in the middle region
Fig 9.19 Glick 3rd Ed

Padlock probes typically have sequences of

approximately 15-20 nucleotides in length, at the 5
and 3 ends that are complementary to the target
sequence and middle region of approximately 50
nucleotides

Although this procedure has not taken off, it is

potentially simpler with fewer steps than OLA
procedure

In addition, this procedure requires one oligonucleotide

as compared to two for OLA

Genotyping with Fluroescence-labled PCR

Primers:

PCR primers labeled with different fluorescent dyes

can be used in development of non-radioactive colorbased detection system

To distinguish between mutant and wild type DNA,

PCR is performed with two different primers

One is exactly complementary to the wild-type DNA

and is labeled at its 5 end with rhodamine (red)

The other is complementary to the mutant DNA and is

labeled at its 5 end with fluorescein (green)

In both cases, amplification is programmed by a third

unlabeled primer that is complementary to the opposite
strand

Since PCR amplification can occur only when the

primer is exactly complementary to target DNA

The presence of these three primers in the same

reaction mixture will result in the amplification of
either the wild type or the mutant DNA or both

depending on which DNAs are initially present

to act as PCR template

If individual is homozygous for the wild type DNA,

after PCR and removal of unincorporated primer the
reaction mixture will fluoresce red

If she/he is homozygous for the mutant DNA, the

reaction mixture will fluoresce green and

if she/he has both mutant and wild type DNA i.e.

hetrozygous, the reaction mixture will fluoresce yellow

Fig 9. 20 Glick 3rd Ed

Mutation at Different Sites within One Gene:

All genetic diseases are not due to a change in single

specific nucleotide within gene

In most cases, a variety of different intragenic sites can

mutate and

Each mutation can cause the same form of the genetic

disease e.g.

-thalassemia; a genetic disease due to a loss in

activity of -globin

Heterozygotes i.e. carries tend to have only mild form

of anemia

In contrast, people who are homozygous for one of

eight or more possible mutations must receive regular
blood transfusion and other treatment to survive

Testing for a change at only one particular nucleotide

site is insufficient

At least eight separate tests are necessary

Although this is feasible but it is costly

Therefore, PCR / Hybridization strategy uses one

reaction assay system has been devised to screen for
mutant nucleotide sites at different location within a
single gene

A set of sequence of specific oligonucleotide probes

(each 20mer) is synthesized

Each oligonucleotide can form a perfect match with a

segment of the target gene that corresponds to the site
of known mutation

A
thymidine
homopolymer
[poly(dT)]
tail
approximately 400 nucleotides long is added to the 3
end of each of the probes

The tail enables the probe DNA to be physically bound

to a predesignated discrete spot on a nylon filter while
ensuring that the rest of the probe is accessible for
hybridization
Fig 9.12, Glick 2nd Ed