Forensic Technology

Polymerase
Chain Reaction
Technology and its applications

he enormous advances a series of polymerization cycles. yielding single strands of

T made in our understanding

of the human genome (and
that of many other species),
would not have been possible, were
it not for the remarkable simple
Each cycle includes three steps:

• Heating step at 910–970C

for 20–30 seconds, where
the DNA template duplex is
and yet exquisitely adaptable denatured to single strands.
technique which is PCR. It involves It causes DNA melting of the
the principle of reactions as - PCR DNA template by disrupting
represents a cyclic reaction where the hydrogen bonds between
target DNA is amplified in vitro by complementary bases,
DNA. For multiple copies of
DNA molecules, the melting
temperature (Tm) is defined as
the temperature at which half
of the DNA strands are in the
double-helical state and half
are in the “random-coil” states.
The melting temperature
depends on both the length
of the molecule, and the

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Polymerase Chain Reaction (PCR) Technology and its applications- Prof. M. Suhail.indd 26 6/24/2010 7:02:35 PM
 Forensic Technology
Polymerase chain reaction (PCR) is a technique widely used in molecular biology,
biotechnology, microbiology, genetics, diagnostics, clinical laboratories, forensic
science, environmental science, hereditary studies, paternity testing, and many
other applications. It is a primer-mediated enzymatic amplification process of
specifically cloned or genomic DNA sequences, which was invented by Dr. Kary
Banks Mullis who received a Nobel Prize in chemistry in 1993, for his invention of
the polymerase chain reaction (PCR). This process, which Kary conceptualized
in 1983, is hailed as one of the monumental scientific techniques of the twentieth
century. It is a method of amplifying DNA; PCR multiplies a single, microscopic
strand of the genetic material billions of times within hours. This technology has
been developed in areas as diverse as criminal forensic investigations, food
science, ecological field studies, and diagnostic medicine.

specific nucleotide sequence dATP (deoxyadenosine temperature step (called hold)

composition of that molecule. triphosphate), dCTP at a high temperature (>900C),
• Annealing step usually at (deoxycytidine triphosphate), and followed by one hold at the
400–650C for 20–40 seconds dGTP (deoxyguanosine end for final product extension or
allowing annealing of the triphosphate) and dTTP brief storage. The temperatures
primers to the single-stranded (deoxythymidine
DNA template. Typically the triphosphate), are bound
annealing temperature is to the free 3’-hydroxyl
about 3-50C below the Tm of end of the new strand.
the primers used. Stable DNA- Only deoxynucleotide
DNA hydrogen bonds are monophosphate is
only formed when the primer incorporated in the DNA
sequence very closely matches chain, cleaving off a
the template sequence. The pyrophasphate group. As
polymerase binds to the PCR progresses, the DNA
primer-template hybrid and generated is itself used as
begins DNA synthesis, and a template for replication,
• Extension step at 680–730C setting in motion a chain
where thermostable DNA reaction in which the DNA
polymerase catalyzes the template is exponentially
synthesis of a new DNA amplified (Fig. 1).
strand by elongation of
the primed strand. The The PCR usually consists
reaction requires two short of a series of 20-40 repeated
oligonucleotides (primers) temperature changes called
flanking the target region to be cycles; each cycle typically
amplified, which are present consists of 2-3 discrete Fig. 1: Showing three fundamental steps of
in large molar excess and temperature steps. Most PCR cycle
hybridize to complementary commonly PCR is carried
segments of DNA. During the out with cycles that have three used and the length of time they
reaction, deoxynucleotide temperature steps. The cycling are applied in each cycle depend
triphosphates (dNTP), i.e., is often preceded by a single on a variety of parameters. These

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Polymerase Chain Reaction (PCR) Technology and its applications- Prof. M. Suhail.indd 27 6/24/2010 7:02:37 PM
 Forensic Technology
include the enzyme used for above 65—700C during the first PCR must be performed in
DNA synthesis, the concentration heating ramp to denaturation at vessels that are compatible with
of divalent ions and dNTPs 940C. Alternatively, an inactive low amounts of enzyme and
(deoxynucleoside triphosphates) form of the enzyme AmpliTaq Gold nucleic acids and that have good
in the reaction, and the melting can be added to all reactions to thermal transfer characteristics.
temperature (Tm) of the primers. prevent misprimed extensions. Typically, polypropylene is used
Adding a pre-PCR heat step at for PCR vessels and conventional,
The target copies are double- 92-950C for 9-12 min synchronously thick-walled microcentrifuge tubes
stranded and bounded by reactivates the enzyme and are chosen for many thermal
annealing sites of the incorporated achieves an “invisible” hot start. In cycler systems. PCR is most often
primers. The 3’ end of the primer both cases, the lowest temperature performed at a 10-100 μL reaction
should complement the target experienced by the reaction scale and requires the prevention
exactly, but the 5’ end can actually components is the stringent primer of the evaporation/condensation
be a non-complementary tail with annealing temperature, assuring processes in the closed reaction
restriction enzyme and promoter best specificity. tube during thermal cycling.
sites that will also be incorporated.
As the cycles proceed, both the AmpliTaq DNA Polymerase is a Mineral oil overlay or wax layer
original template and the amplified highly characterized recombinant serves this purpose. More recently,
targets serve as substrates for the enzyme for PCR. It is produced in E. 0.2 mL thin-walled vessels have
denaturation, primer annealing, coli from the Taq DNA polymerase been optimized for the PCR process
and primer extension processes. gene, thereby assuring high purity. and oil-free thermal cyclers have
Since every cycle theoretically It is commonly supplied and used been designed that use a heated
doubles the amount of target as a 5 U/μL solution in buffered cover over the tubes held within the
copies, a geometric amplification 50% glycerol. sample block. In a standard aliquot
occurs. PCR is conceptualized of Taq DNA polymerase used for a
as a process that begins when The enzyme is a 94-kDa protein 100μL reaction, there are about 1010
thermal cycling ensues. with a 5’-3’ polymerization activity molecules. Each PCR sample should
that is most efficient in the 70- be evaluated for the number of target
The annealing temperature 800C range. This enzyme is very copies it contains or may contain.
sets the specificity of the reaction, thermostable, with a half-life at For example 1ng of λ DNA contains
assuring that the primary primer 950C of 35-40 min. AmpliTaq DNA 1.8 x 107 copies. For low input copy
binding events are the ones polymerase requires magnesium number PCR, the enzyme is in great
specific for the target in question. ion as a cofactor and catalyzes excess in early cycles.
In preparing a PCR reaction on ice the extension reaction of a primed
or at room temperature, however, template at 720C. As mentioned As the amplicon accumulates in
the reactants are all present for above, the four dNTPs (consisting later cycles, the enzyme becomes
nonspecific primer annealing to of dATP, dCTP, dGTP, and dTTP limiting and it may be necessary
any single-stranded DNA present. or dUTP) are used according to to give the extension process
Since Taq DNA polymerase has the base-pairing rule to extend incrementally more time. Thermal
some residual activity even at the primer and thereby to copy cyclers can reliably perform this
lower temperatures, it is possible the target sequence. Modified automatic segment extension
to extend these misprimed hybrids nucleotides (ddNTPs, biotin-11 procedure in order to maximize
and begin the PCR process at the -dNTP, dUTP, deaza-dGTP, and PCR yield.
wrong sites. By withholding a key fluorescently labeled dNTPs)
reaction component, such as Taq can be incorporated into PCR The Polymerase Chain Reaction
DNA polymerase, until an elevated products. The PCR buffer for (PCR) Technology can be applied
temperature can be reached, Taq DNA polymerase consists of in various forms needing small
the possibility of mispriming is 50 mM KC1 and 10 mM Tris-HCl, modification to be made to the
avoided. pH 8.3, at room temperature. This standard PCR protocol to achieve
buffer provides the ionic strength a desired goal. The prominent
This can be accomplished and buffering capacity needed categories with their applications
by a manual addition of enzyme during the reaction. are described below.

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 Forensic Technology
PCR Analysis of DNA from the simultaneous employment of an polymerase chain reaction (qPCR)
Fresh and Decomposed amylase assay. to detect RNA viruses has become
Bodies and Skeletal increasingly important as a
Remains in Medico-legal Mutation and prognostic marker and in patient
Death Investigations Polymorphism Detection management, for example, in human
One of the greatest values of Mutational analysis has many immunodeficiency virus (HIV) and
polymerase chain reaction (PCR) for applications, for example, markers hepatitis C virus (HCV) infection.
the death investigator lies in the fact of disease, cancer research, Drug therapies can be monitored
that even minute amounts of DNA and genotyping. Mutations are by regularly checking viral load,
or extensively damaged (degraded) responsible for diseases, such as indicating whether the regime is
DNA can be successfully amplified sickle cell anemia, cystic fibrosis, sufficient, or whether alternatives
and thus become amenable for and adrenal leukodystrophy. An should be sought. It is therefore
typing procedures. expansion of the CAG repeat in crucial that the systems used are
exon 1 of the androgen receptor ultrasensitive and give accurate and
In medico-legal death alone can result in Kennedy’s reproducible results.
investigations, the types of DNA disease, spinocerebellar ataxia,
recovered from a body can be or fragile X syndrome. Therefore, Single-Locus and Multilocus
divided into two areas: DNA polymorphism and mutational VNTR-PCR
evidence, which adheres to the analysis are of use in diagnosing Nonisotopic probes have
surface of the body or is present these diseases. been widely adopted for DNA
within a body cavity (e.g., blood, fingerprinting and DNA profiling
semen, saliva, nasal secretions, Detection of polymorphism because of their ease and speed
hairs, shed scalp skin, urine, faeces); and mutational analysis is also of use and obvious safety and
and biological material, which widely used in the field of cancer environmental advantages.
belongs to the deceased (e.g., research. Mutation detection has Nonisotopic DNA probes designed
liquid blood, soft tissues, bones, demonstrated that p53 function to detect variable number of tandem-
teeth, fingernails) and can be used is lost in approximately 50% of all repeat (VNTR) sequences are
as reference samples. In general, cancers and that the loss of function typically single-stranded oligomers
the success of DNA profiling is caused by point mutations. It of 20-30 nucleotides, with sequence
depends most on the environmental also demonstrates that a mutation complementary to the target
conditions the body was exposed to in BRCA 1 or BRCA 2 increases tandem-repeat sequence. There
and on the proper preservation and susceptibility to breast and ovarian are two types of probes used for
collecting procedures. cancer. Microsatellite analysis is the analysis of VNTR sequences:
used in cancer research to study multilocus probes (MLP) and single-
PCR Analysis from LOH (loss of heterozygosity) and locus probes (SLP).
Cigarette Butts, Postage microsatellite instability. LOH
Stamps, Envelope Sealing studies have demonstrated most MLPs consist of tandem
Flaps, and Other Saliva- genetic alterations that occur in repeats containing a minisatellite
Stained Material bladder cancer are on chromosome “core” sequence that can
The polymerase chain reaction 9 and that microsatellite instability simultaneously detect a number
(PCR) has offered the forensic is commonly found in colorectal of highly polymorphic loci to
scientist a new range of sensitivity in cancer. generate individual-specific DNA
the examination of forensic samples. “fingerprints”. These probes have
PCR has been successfully used In summary, polymorphism and found a number of applications in
to amplify specific DNA fragments mutational analysis is a major tool in the field of identity analysis, including
from extremely small amounts of science today, without which much of forensic and paternity testing and
DNA present on cigarette butts, our current understanding of genetics cell-line verification.
postage stamps, envelope sealing would not have been possible.
flaps, and other saliva-stained When hybridized to Southern
materials. In addition to DNA typing Ultrasensitive Quantitative blots under conditions of low
results, it is at times desirable to PCR to Detect RNA Viruses stringency, each multilocus probe will
confirm the presence of saliva by The use of quantitative detect a family of minisatellites that

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 Forensic Technology
all share the same “core” sequence. mtDNA existing in hundreds if not
This produces the multiband DNA thousands of copies per cell.
“fingerprint” pattern. Several different
MLPs have been isolated, and most The discriminatory power of
can be used to detect minisatellites in mtDNA testing arises from the
a wide range of species. PCR-VNTRs polymorphic nature (between
are important markers for questions unrelated individuals) of the two
of identification, individualization, hypervariable regions (HV1 and
and discrimination. They already HV2) located within the D-loop of
form an integral part of forensic the mtDNA genome. The haploid,
DNA analysis. maternal inheritance patterns of
mtDNA transmission between
Direct and Indirect In Situ generations, allow an inclusion or
PCR exclusion to be made when the
In recent years, the development sample sequence is compared
of in situ technologies has made to that of a maternal reference.
good progress. In situ hybridization Related individuals will share
(ISH) has become an important tool similar polymorphisms relative to a
and has enabled the pathologist to consensus standard.
demonstrate infectious pathogens
or mRNAs in tissue sections or Reverse-transcriptase-
cytospins without destruction of polymerase chain reaction
morphology, thus enabling the (RT-PCR) in Biomedicine
assignment of signals to individual RT-PCR has become one of the
cells or cell compartments. most widely applied techniques
in biomedical research. The ease
PCR has become an important with which the technique permits
diagnostic as well as research tool in specific mRNA to be detected and
molecular biology, clinical chemistry, quantified has been a major asset
and pathology. With the invention in the molecular investigation of
of IS-PCR, the amplification power disease pathogenesis. Disease-
of solution-phase PCR with no related imbalances in the
limitations in the amount of template expression of specific mRNAs can
was hoped to be transferred to the in be sensitively and quantitatively
situ techniques. determined by RT-PCR. RT-PCR
also offers many opportunities
Amplification and in diagnostics, allowing sensitive
Sequencing of detection of RNA viruses such as
Mitochondrial DNA in Human Immunodeficiency Virus
Forensic Casework (HIV) and Hepatitis C Virus (HCV).
Mitochondrial DNA (mtDNA)
typing is increasingly used for the RT-PCR is an integral component
forensic identification of human of many methodologies that are
remains. This is especially true essential to biomedical research,
when only limited quantities of including in situ localization of mRNA,
sample are present, such as antibody engineering, and cDNA
when the sample has undergone cloning. The greatest advantage
extensive degradation and nuclear- of RT-PCR in the analysis of mRNA
typing methods are ineffectual One is its extraordinary sensitivity.
characteristic of mtDNA responsible Using nested RT-PCR, mRNA can
for the increasing reliance is the high essentially be detected at the level
copy number of mtDNA per cell, with of single copies. The capability
32
Polymerase Chain Reaction (PCR) Technology and its applications- Prof. M. Suhail.indd 32
for such sensitive detection and
analysis of mRNA is an enormous
asset in many research areas,
such as developmental biology.
Another area that requires analysis
of small local populations of cells
is brain research. Understanding
the function of the brain is one of
the greatest challenges in biology
today. Research in brain biology will
benefit significantly from RT-PCR;
the expression of low-abundance
mRNAs can now be measured in
small tissue samples from specific
areas of the brain.
Identification and
Differentiation of Brucella
abortus Field and Vaccine
Strains by BaSS-PCR
Brucellosis is a bacterial disease
affecting livestock worldwide. Only
one assay, the AMOS assay (named
for the species it identifies: B.
abortus, B. melitensis, B. ovis, and B.
suis), has been developed to identify
and differentiate the major Brucella
species and also to differentiate the
B. abortus vaccine strains from field
isolates. The AMOS assay is a single-
tube multiplex PCR assay designed
to amplify up to three independent
targets differing in size. Identification
is based on the pattern of DNA
products amplified from specific DNA
targets located within the unknown
isolate’s genome.
PCR Technology and
Applications to Zoonotic
Food-Borne Bacterial
Pathogens
PCR testing offers the
possibility to improve detection
and characterization of pathogenic
bacteria, since one can target
species-specific DNA regions and
specific traits of pathogenicity,
especially genes coding for
toxins, virulence factors, or major
antigens. The PCR technique
has several advantages over
May-June 2010  Medical Equipment & Automation
6/24/2010 7:02:39 PM
 Forensic Technology
classical bacteriology with respect Y, hepatitis A virus (HAV), hepatitis that is the causative agent of
to detection limit, speed, and E virus , poliovirus , and coxsackie toxoplasmosis in humans and
potential for automation. The latter B2 virus. animals. It is responsible for
capability is indeed necessary for abortion and congenital defects
application of the test in extensive Specificity and in humans and is an important
screening programs. Currently, Performance of Diagnostic cause of abortion in domestic
probes and PCR methods are PCR Assays livestock, especially sheep, goats,
available for many important The undisputed success & pigs. Direct detection of T. gondii
food-borne pathogens, such as of detection assays based on parasites by amplification of DNA
Salmonella, enterohemorrhagic the polymerase chain reaction using polymerase chain reaction
E. coli, Yersinia enterocolitica, (PCR) has been largely due to its (PCR) has, therefore, become
Campylobacter spp., and Listeria rapidity in comparison to many a valuable asset. Independent
monocytogenes. conventional diagnostic methods. studies show that B1 locus is the
For instance, detection and best target identified so far for the
Long PCR Amplification of identification of mycobacteria, routine & sensitive detection of T.
Large Fragments of Viral chlamydiae, mycoplasmas, gondii in human and animal tissue.
Genomes brucellae, and other slow-growing
Long PCR has also been used to bacteria can be accelerated from Sex Determination by
amplify the complete mitochondrial several days to a single working PCR Analysis of the X-Y
genome and large genomic day when clinical samples are Amelogenin Gene
fragments for the determination directly examined. Among known X-Y homologous
of deletions in genetic diseases genes, the amelogenin gene is
or gene fusions in cancer. Other Other microbial agents that the most suitable for the sex test
applications in microbiology are difficult to propagate outside by PCR: Following a single PCR
include bacterial typing and their natural host often remain with one pair of primers, X- and
amplification of Plasmodium undetected by techniques relying Y-specific products with different
falciparum DNA. Long PCR has on cultural enrichment, thus sizes are simultaneously detected
been applied with great success rendering PCR the only viable because of difference in the lengths
to viral genomes. For example, alternative to demonstrate their of corresponding introns. However,
amplification of full-length, near presence. Additionally, there is the sensitivity of amplification of this
full-length, or large fragments of the enormous potential of DNA single-copy gene is relatively low.
the genome of many DNA viruses amplification assays with regard Then downsizing of PCR products
has been achieved, such as for the to sensitivity and specificity. with the use of a different set of
hepatitis B virus (HBV), the human primers and nested PCR technique
papilloma virus, SV-40, varicella- Toxoplasma gondii is an has been applied to improve
zoster virus , the proviruses of the important intracellular protozoan the sensitivity. 
simian foamy virus of chimpanzees
(SFVcpz), human T-cell leukemia
Prof. Mohammad Suhail, M.Sc. M.Phil. PhD is Hon. Director, City
virus 1 (HTLV-1), and human Nursing & Maternity Home Research Center, Minhajpur, Allahabad has
immunodefciency virus type 1 (HIV- established Department of Proteogene Life Sciences to work as its
1) and type 2 (HIV-2). Director. He served at University of Allahabad as Reader & Head and
Professor & Head, Department of Biochemistry. He has been the visiting
Scientist under Teachers’ Exchange Program by the Italian Education
Long RT-PCR for RNA viruses Ministry. His specialization is in Clinical Biochemistry & Molecular
has also been successful. Large Biology. He has completed three Research Projects funded by Indian
Prof. Mohammad
fragments of the viral genome have Suhail
Council of Medical Research, UGC, New Delhi & Council of Science and
been amplified for the tick-borne Technology, U.P. Several PhD, Master of Surgery, Doctor of Medicine
degrees were awarded under his guidance from University of Allahabad
encephalitis virus, the Norwalk- & its affiliated Medical College. Has chaired the 51st. Annual Meeting
like viruses , the hepatitis C virus of the Society of Biological Chemists (India). He is the member of New
(HCV), and the bovine torovirus . York Academy of Sciences, and Life Member of National Academy
The near full-length genome was of Sciences, India. He has served as expert in various academic &
administrative boards and is the Reviewer of National & International
amplified for HCV and the full- Journals.
length genome for the potato virus

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Polymerase Chain Reaction (PCR) Technology and its applications- Prof. M. Suhail.indd 35 6/24/2010 7:02:39 PM