Polymerase Chain Reaction, Types and
Applications (PCR)
Polymerase Chain Reaction is a technique used to amplify a specific region of a DNA molecule
to generate multiple copies. This innovative technology was developed by American
biotechnologist Kary Mullis in the year 1983. He was awarded with noble prize for his innovative
work in 1993 (Singh et al., 2014). The important component of PCR include:
Template DNA: Target DNA molecule to be amplified.
Taq polymerase: A DNA polymerase enzyme isolated from bacterium ‘Thermus
aquaticus’ used for replication process. It is a thermostable enzyme which can sustain
its activity at a wide range of temperatures.
Oligonucleotide primers: A short sequence of DNA which are complementary to the
template DNA and serve as a DNA synthesis starting point. These are designed
specifically for the amplification of region of interest.
Deoxyribonucleotide triphosphates (dNTPs): Are the building blocks required for
replication of DNA. Adenine (A), guanine (G), cytosine (C), and thymine (T) are the four
different dNTPs used in the reaction. DNA polymerase adds each complementary base
to the new growing DNA strand according to the original template.
PCR buffer: The reaction mixture contains the buffer which provide the optimum pH
for the PCR. Magnesium is another important constituent of the PCR cocktail which acts
as a cofactor regulating the activity of Taq DNA polymerase.
PCR has Three Main Steps:
Denaturation:The double stranded target DNA is heated to unwind into two single
strands. Both the strands serve as templates. 95
0C is usually an optimum temperature
for denaturation.
Annealing: Primers anneal to the template strand by lowering temperature. Annealing
temperature (50-65
0C) is the most critical for the proper amplification and depends upon
the GC content of primers.
Extending: Temperature is raised (usually to 72
0C), and the new strand of DNA is
synthesized from 5’ to 3’ end by the Taq polymerase enzyme.
Types of PCR:
Reverse transcriptase PCR (RT- PCR): This technique allows the detection of RNA in the sample. The RNA molecule is
reverse transcribed into complementary DNA (cDNA) using the enzyme reverse
transcriptase. This is followed by the amplification of cDNA using standard PCR.
Real time-PCR or quantitative PCR (qPCR): Detects fluorescent reporter dyes like SYBR Green, used to quantify DNA amplification
at each PCR cycle. The fluorescence signal increases proportionally to the amount of
replicated DNA and hence the DNA is quantified in “real time”. The fluorescence grows
to a point where it becomes quantifiable during the log linear phase of amplification,
which is known as the Threshold cycle (CT) (Singh et al., 2014).
RT-PCR/qPCR combined: This involves the quantitative detection of RNA expression using both RT-PCR for
cDNA synthesis and q-PCR for real time amplification.
PCR Modifications:
Asymmetric PCR amplifies only one strand of the target DNA molecule by using
unequal primer concentrations. The technique is mainly applied in sequencing or
hybridization probing where only one strand of DNA is required.
Colony PCR R rapidly screens the colonies of bacteria or yeast that are grown on the
selective media after cloning to verify whether segment of interest is successfully
transformed or to amplify the section of insert.
Degenerate PCR amplifies the unknown DNA sequences, mainly coding gene
sequences using degenerate set of primers. The primers are constructed based on
the known sequences of gene homologs.
Hotstart PCR is as good as conventional PCR, only the Taq polymerase is added
after other components are heated to DNA denaturation temperature. This avoids
the nonspecific amplification and prevents mis-priming and primer dimer formation.
Inverse PCR amplifies DNA with only one known sequence. It is used to determine
the location of the insert
Multiplex PCR is used for the simultaneous amplification multiple targets in a
single reaction with a specific set of primers pair of each target. Two or more probes
that can be distinguished from each other and detected simultaneously using this
technique.
Nested PCR involves the two consecutive amplification reactions with two distinct
primer set. The first amplification reaction product serves as a template for second
reaction. This variant of PCR minimizes the non-specific amplification and increases
the sensitivity and specificity of PCR.
Touchdown PCR (TD-PCR) s a modification of PCR in which the initial annealing
temperature is higher than the optimal Tm of the primers and is gradually reduced
over subsequent cycles until the Tm temperature or “touchdown temperature” is
reached. This is used to increase the specificity of PCRs.
Amplification refractory mutation system (ARMS) PCR is used to detect a
single base change or SNP using sequence-specific primers. Here two different set
of primers are constructed, mutant and wild type.
The 3’ end each primer is modified so that normal primer can amplify only normal allele
and mutant primer can amplify only mutant allele.
Multiplex ligation-dependent probe amplification (MLPA) allows amplification
of multiple targets using the single pair. It’s used for the molecular detection of
variation in copy numbers. It serves as an important molecular diagnostic tool for
identification of genetic diseases.
Applications of PCR
1. PCR is highly advantageous in forensic medicine where it identifies an individual
from million others. The DNA extracted from the crime scene blood/tissue sample
is amplified and compared with other suspects or a DNA database and the convict
is found. DNA fingerprinting can also be used for parental testing in order to
determine a child’s biological parentage (Singh et al., 2014).
2. The polymerase chain reaction (PCR) is a fantastic diagnostic tool. It’s highly
useful for identification of genetic diseases. PCR can detect locations, sizes, and
natures of harmful mutations.
3. HLA typing is performed using PCR prior to organ transplantation, to determine
donor-recipient compatibility.
4. PCR has huge application in identification of infectious diseases (viral, bacterial,
parasitic, etc.). It can produce reliable results rapidly from a minute biological
specimen (Kadri, 2020).
References:
Kadri, K., 2020. Polymerase Chain Reaction (PCR): Principle and Applications, in:
L. Nagpal, M., Boldura, O.-M., Baltă, C., Enany, S. (Eds.), Synthetic Biology – New
Interdisciplinary Science. IntechOpen. https://doi.org/10.5772/intechopen.86491
Kadri, K., 2020. Polymerase Chain Reaction (PCR): Principle and Applications, in:
L. Nagpal, M., Boldura, O.-M., Baltă, C., Enany, S. (Eds.), Synthetic Biology – New
Interdisciplinary Science. IntechOpen. https://doi.org/10.5772/intechopen.86491
Singh, J., Birbian, N., Sinha, S., Goswami, A., 2014. A critical review on PCR, its
types and applications 17.
