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POLYMERASE CHAIN REACTION AND

Wg Cdr PK MENON•,Lt Col K KAPILA *,Brig VC OHRI #

ABSTRACT
The use of Polymerase Chain Reaction (PCR) in infectious disease diagnosis, has resulted in an ability to diagnose early and treat
appropriately diseases due to fastidious pathogens, determine the antimicrobial susceptibility of slow growing organisms, and
ascertain the quantum of infection. This article outlines the PCR, some of its modifications and their application in infectious disease
diagnosis.
MJAFI 1999; 55 : 229-231
KEY WORDS: Microbial diagnosis; PCR.

Introduction: stranded DNA called primers are then chemically syn-

T
he study of genetic structure and function re- thesised. The primers are complimentary to the se-
quires a sufficient amount of DNA, which was quences flanking the unique region. The concentration
synthesised using a difficult process involving of the primer is several million folds than the target
bacteria. A surprisingly simple method for making un- DNA. Two primers are used, one designed to anneal
limited copies of DNA fragments was conceived by to the sense strand, the other designed to anneal to the
Kary Mullis in 1983 during a moonlit drive through anti sense strand. The primers are mixed with a buff-
the mountains pf Northern California's redwood coun- ered solution of template DNA, nucleotides (dNTPs),
try. The polymerase chain reaction (PCR) is a tech- magnesium chloride and DNA polymerase [2]
nique for copying a piece of DNA in the laboratory Earlier DNA polymerases were denatured by heat
with readily available reagents. With each step of the and required replenishment after every denaturing cy-
reaction the number of DNA molecules increases ex- cle. A thermostable DNA polymerase called Taq p0-
ponentially and in a few hours of running the reaction lymerase is derived from the bacillus Thennus acqua-
more than 100 billion copies can be made which can ticus (found in hot spring). These thermostable DNA
be easily detected. The PCR has progressed to become polymerases have revolutionised PCR technology and
an important tool in the rapid diagnosis of infectious enable the reaction to be carried out by a single addi-
disease [1]. tion of Taq polymerase [3].
Watson and Crick in 1953 suggested the double The double stranded template DNA is denatured by
stranded DNA helical structure, the base adenine (A) heating to a temperature above its melting (98°C) and
binding to thymine (T) and guanine (0) to cytosine the strands of DNA separate. The temperature is low-
(C). On raising the temperature the strands separate ered so as to allow annealing of the primer to the
(melt), and lower temperatures cause them to rejoin template DNA. The Taq DNA polymerase extends the
(anneal), with the nucleotides realigning to regain the primer if it has bound to the target template strand of
double helical structure. The unique sequences of DNA by adding appropriate complementary nucIeo-
DNA molecules coding for the pathogen are detected tides to the three prime [3'] end of the bound primer
by PCR. This is an easier and more rapid technique (Fig 1). High primer concentration ensures that primer
than traditional exacting methods for cultivation and annealing and extension are preferred over template
identification of fastidious pathogens. strand reannealing. The reaction is stopped by raising
the temperature and strand denaturation occurs. The
Polymerase chain reaction temperature is then lowered to permit primer anneal-
To detect a pathogen by the PCR reaction it is es- ing, and the cycle is repeated. The PCR reaction is
sential to know the sequence of nucleotides flanking represented diagrammatically in Fig 1.
an unique region on its DNA. Short strands of single

* Readers. Depanment of Microbiology. Anned Forces Medical College. Pune 40, 1/ Commandant, 167 Military Hospital. C/o 56 APO.
230 Menon, Kapila and Ohri
Double str..ded
5' ....etONA 3' recombinant modified form of Thennus thennophilius
3' 5' DNA polymerase gene expressed in E.coli, the en-
zyme rTth, has an efficient reverse transcriptase activ-
ity in the presence of manganese, and DNA polym-
erase activity in presence of magnesium. Use of this
enzyme has enabled the determination of cellular

_
mRNA expression by a single step procedure [7]. The

-
5' . importance of RT PCR lies in its ability to detect RNA
Viruses (e.g. HIV), study intracellular signals (e.g. in-
tracellular interleukin expression) and therapeutic to
quantify viral loads (e.g. semi quantitative RT PCR for
Fig. I: Shows the amplification of target DNA by peR using two HIV loads to monitor response to anti retroviral ther-
DNA primers. Taq polymerase and nucleotides. The re- apy).
petitive cycle of deanturation, annealing, extention results
in two copies of the target DNA, which is now available Nucleic acid sequence based amplification
for the next cycle of amplification. resulting in a logarith- (NASBA) : This is an ingenious method which is iso-
mic increase in DNA copies after each cycle. thermal and does not require thermal cycling. RNA is
converted to cDNA which carries a n RNA polym-
After the firSt three cycles the majority of the DNA
erase sequence at one end in the initiation phase and
strands would be those which are bound by the prim-
the adherent RNA is removed by RNase H. The Re-
ers at both ends. Further amplification cycles result in
verse transcriptase (RT) then syn.hesises a comple-
exponential increase of the amplified DNA and thus
mentary DNA molecule to acquire a double stranded
would be of a defined length. The reaction is carried
structure. The n RNA polymera;~e then synthesises
out as a 50 ul volume reaction mixture. The product is
large number of RNA copies. The cycle is repeated to
electrophoresed on an agarose gel. On staining with
produce antisense RNA as the final amplified product
ethidium bromide and visualisation in UV light the
[8].
DNA molecules fluoresce. Molecular weight measure-
ment is carried out by comparing migration with Ligase chain reacrion(LCR) : This uses four prim-
markers of known molecular weight [2]. ers so designed that on annealing the primers bind
such that they are immediately adjacent and com-
Specific methods for nucleic acid amplification pletely cover the target sequence. The DNA ligase
reactions present joins both the fragments, which can then be
Some important modifications of the PCR are: detected [9].
Multiplex peR In multiplex PCR simultaneous am- Applications of the peR in Infectious disease
plification of more than one genetic locus, using more diagnosis
than one set of primers is carried out in the same reac-
tion. This can be used to differentiate between eti- Tuberculosis : With increasing incidence of both
ological agents responsible for a lesion. Depending on HIV infection and multi drug resistant strains of M.tu-
the molecular ,weight of the fragment amplified the berculosis. early detection is vital for diagnosis and
etiologic agent can be determined [4]. therapy. Classical techniques for detection of infection
have a drawback in that the organism is fastidious and
Randomly amplified polymorphic DNA (RAPD) grows slowly. Many molecular strategies for detection
typing: This is used for epidemiological identification of mycobacteria have been developed. Important ones
of bacterial isolates. One or more short primers of are PCR, transcription mediated amplification, nucleic
variable length are arbitrarily selected, and allowed to acid sequence based amplification and ligase chain re-
anneal to the template DNA at a low stringency. PCR action [10]. Mycobacterial speciation which is time
amplification is carried out and the products resolved consuming and exacting by conventional techniques,
electrophoretically to yield DNA fingerprints which can be differentiated relatively easily by Multiplex
differs according to the degree of relatedness of the PCR [4]. Molecular susceptibility testing for first line
strains under investigation [5]. drugs INH and rifampicin is based on the fact that
Reverse Transcriptase (RT) PCR: This technique there is a mutation in the cat gene and the rpoB gene.
uses RNA as a starting template. RNA is converted to This can be detected by a peR amplification of the
cDNA by a retroviral reverse transcriptase. The cDNA gene fragment followed by a simple electrophoresis in
is amplified and the amplified band detected [6]. A denaturing gels for single stranded confirmational
MJAFI. VOL 55. NO.3. 1999
PCR in Infectious Diagnosis 231

polymorphism (SSCP) analysis. This technique is sen- services will result in a challenging and exciting time
sitive and is able to detect even point mutations for the clinical microbiologist.
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