Technology – Labassure

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:

1. Template DNA: Target DNA molecule to be amplified
2. 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.
3. 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.
4. 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.
5. 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:

1. Denaturation: The double stranded target DNA is heated to unwind into two single strands. Both the strands serve as templates. 950C is usually an optimum temperature for denaturation.
2. Annealing: Primers anneal to the template strand by lowering temperature. Annealing temperature (50-650C) is the most critical for the proper amplification and depends upon the GC content of primers.
3. Extending: Temperature is raised (usually to 720C), and the new strand of DNA is synthesized from 5’ to 3’ end by the Taq polymerase enzyme.

Types of PCR:

1. 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.
2. 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).
3. 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:

1. 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.
2. Colony PCR 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.
3. 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.
4. 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.
5. Inverse PCR amplifies DNA with only one known sequence. It is used to determine the location of the insert.
6. 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.
7. 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.
8. Touchdown PCR (TD-PCR) is 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.
9. 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.
10. 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
• Polymerase Chain Reaction (PCR) [WWW Document], n.d.. Genome.gov. URL https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction (accessed 2.18.22).
• Singh, J., Birbian, N., Sinha, S., Goswami, A., 2014. A critical review on PCR, its types and applications 17.

Polymerase Chain Reaction, types and applications (PCR)

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