Different types of PCR
Polymerase chain reaction (PCR) is broadly classified on the basis of slight modifications in the standard PCR process. Different types of PCR are as follows:
- Nested PCR
- Inverse PCR
- Reverse Transcription PCR (RT-PCR)
- Asymmetric PCR
- Quantitative PCR (Q-PCR)
- Quantitative Real-Time PCR (QRT-PCR or RTQ-PCR)
- Touchdown PCR
- Colony PCR
- Allele-Specific PCR
- Polymerase Cycling Assembly (PCA) or Assembly PCR
- Linear-After-The-Exponential PCR (LATE-PCR)
- Dial-Out PCR
- Helicase-dependent amplification
- Hot Start PCR
- Inter-sequence Specific PCR
- Ligation mediated PCR
- Methylation-specific PCR
This technique is proposed to decrease the contaminants in amplified DNA because of random primer binding amplification. Nested PCR is performed with two different sets of primers in two consecutive PCR cycles. The subsequent set is expected to amplify the secondary target inside the product of the primary run. This technique is exceptionally successful, although it requires much more knowledge of the involved sequences.
It is performed when only single internal sequence of template DNA is known. This technique is proper in the identification of the flanking sequences to the various gene inserts. The process includes a sequence of digestion and self-ligation of the template DNA, which results in chunks of DNA with known sequences at the ends of an unknown sequence.
Reverse Transcription PCR (RT-PCR)
It is used to amplify and identify known sequences from RNA libraries. The RNA obtained from the sample is then converted into complementary DNA (cDNA) by the action of enzyme reverse enzyme transcriptase. Typical PCR has performed afterwards. RT-PCR is extensively used in identification and the determination of gene expression.
It selectively amplifies one strand of the template DNA more than the other. It is used in the hybridization probing in which only one strand is considered ideal. Further amplification is achieved by carrying out standard PCR in the presence of excess primers for the chosen strand.
Quantitative PCR (Q-PCR)
This is the indirect procedure of measuring initial amounts of RNA, cDNA or template DNA. Q-PCR is generally used to determine the quantity of PCR product quickly. Q-PCR is also used for the detection of specific sequences inside the DNA sample.
Quantitative Real-Time PCR (QRT-PCR or RTQ-PCR)
This process uses fluorescent dyes to monitor and measure real-time amounts of amplified products. This quantitative real time PCR is used in conjugation with QPCR.
This technique reduces the non-specific primer binding by decreasing the annealing temperature between two consecutive cycles of PCR.
Clones (Ex. E. coli) are analyzed for the correct ligation products. Specific colony is picked through a sterile toothpick and introduced into the master mix. The PCR is operated with extended time at 95oC.
This technique is based on the single nucleotide polymorphism (SNPs), allele-specific PCR is often used for cloning and diagnostic purposes. Some prior knowledge about DNA sequence of alleles and their difference is required. Allele-specific PCR involves the use of primers SNP containing 3′ end. Successful allele-specific PCR amplification and SNP-specific primers indicate the presence of particular SNP in the sequence.
Polymerase Cycling Assembly (PCA) or Assembly PCR
Involves the artificial production of the long DNA sequences in the presence of long oligonucleotides and short overlapping segments following the standard PCR procedure. The long oligonucleotides flip in both directions (sense and anti-sense), the overlapping segment decides the order of PCR fragments, facilitating the selective production of the final long DNA.
Linear-After-The-Exponential PCR (LATE-PCR)
In this technique, the limiting primer (primer present in the lower amount) has a higher melting point than the excess primer (primer present in sufficient amount) for maintaining the reaction efficiency since the concentration of the limiting primer decreases in the middle of the reaction.
It is a method for recovering DNA molecules for the gene synthesis. A library of DNA is modified with specific flanking tags before the sequencing. Later, the tag directed primers allow the recovery of DNA of desired sequences by PCR.
It is related to the traditional PCR, but it uses a constant temperature instead of cycling. DNA helicase is used instead of the thermal denaturation step.
Hot Start PCR
This technique lay off the possibility of non-specific amplification during the initial cycles of PCR. The reaction ingredients are heated at the denaturing temperature that is 95oC before the addition of polymerase. Inhibitors and antibodies are introduced in the reaction mixture to inhibit DNA polymerase activity, which is dissociated at high temperatures.
Inter-sequence Specific PCR
This modification of PCR technique is often used for DNA fingerprinting, which requires the amplification of regions placed between the simple sequences repeats to generate a unique and specific fingerprint/pattern of amplified DNA fragments.
Ligation mediated PCR
It specifically utilizes small linker molecules of DNA (to be inserted) and various primers capable of annealing with the linker DNA. This technique is extensively used for DNA sequencing and foot-printing.
This technique is used for the detection of CpG islands in the genome. In Methylation-specific PCR, DNA is treated with sodium bisulfate, which converts the unmethylated cytosine base into uracil, which is identified/recognized by the PCR primer thymine bases. Two different PCR sets are carried out on the altered DNA using identical primer sets except for the primers’ CpG islands. The primer sets recognize the cytosine bases and other sets of primers recognize the uracil to amplify the unmethylated DNA. Q-PCR can also be performed to know the quantitative information regarding methylation.
Conceptual MCQs on Different types of PCR (solved)
Question 1: A researcher is trying to alter five successive base pairs in the middle of a PCR amplified DNA fragments. Which technique is the most recommended for performing such experiment?
A- DNA ligation
B- Electrophoretic mobility shift assay (EMSA)
C- PCR mutagenesis
D- Restriction enzyme digestion
Correct Answer: Option C PCR mutagenesis
The most recommended technique for such type of modifications is PCR mutagenesis. It can synthesize primers that bind imperfectly at the desired site of mutagenesis. These primers will now contain the new 5-base pair sequence to be introduced in the new DNA strand. Firstly, the PCR will amplify the DNA fragment containing the mutant sequence and the upstream sequences. The second cycle of PCR will amplify the new DNA strand containing the mutant sequence and also the downstream sequences. And the final cycle of PCR will amplify one DNA fragment from the two templates, using the original primers to amplify the full-length DNA strand.
Question 2: Polymerase chain reaction (PCR) utilizes a heat-stable polymerase (Taq polymerase) to amplify DNA strand. Which of the statement best describes why thermo-stable polymerases are required for PCR?
A- PCR needs thermal cycling, and the Taq polymerases can be inactivated since they are not needed during the low-temperature steps.
B- Heat stable (Taq) polymerase do not break the dsDNA unless the temperature is very high. Therefore a heat-stable (Taq) polymerase is required.
C- PCR needs thermal cycling, and the heat-stable (Taq) polymerases will not denature to lose efficacy in DNA polymerization during the high temperature (95oC) step.
D- The interaction between divalent cations and DNA polymerase is much efficient during high-temperature steps. Thus PCR requires heat-stable DNA polymerase.
E- Heat stable (Taq) polymerases are cheaper than standard polymerases; thus, they are more suitable for amplifying DNA.
Correct Answer: Option C
PCR needs thermal cycling, and the heat-stable (Taq) polymerases will not denature to lose efficacy in DNA polymerization during the high temperature (95oC) step. This makes it unique among Different types of PCR.
Polymerases often denature at higher temperatures. If a standard polymerase were used, it would denature during high temperature (denaturation) step, which is needed for breaking the double-stranded DNA into single-stranded DNA templates. By using this heat-stable (Taq) polymerase, the enzyme will not denature. The DNA strands will continue to amplify by the Taq polymerase added at the start of PCR.
Question 3: What is the correct order of the three steps of PCR?
A- Extension , denaturation, annealing
B- Denaturation, annealing, extension
C- Annealing, extension, denaturation
D- Denaturation, extension, annealing
Correct Answer: Option B
Denaturation, annealing, extension
The steps of PCR involve denaturation, annealing and extension.
Denaturation is done at a temperature around 94-98°C to break hydrogen bonds of double-stranded DNA.
Annealing is the second step of PCR completed at 50-65°C. The annealing step must be performed at a low temperature for the attachment of primers to the single strand, but high enough to avoid unspecific hybridization.
The extension or elongation step of PCR allows the DNA polymerase to synthesize the template DNA strand’s latest copy. The optimum temperature depends on the DNA polymerase being used but usually ranges from 75-80°C.
Question 4: Which of the following is/are not a component of a polymerase chain reaction mixture?
A- Buffer solution
B- DNA polymerase
D- DNA template strand
Correct Answer: Option C
The ingredients of PCR are DNA (Taq) polymerase, buffer, primers, magnesium chloride, dNTPs (adenine, guanine, cytosine, thymine), deoxynucleotide triphosphates and the DNA template strand.
In this article we have discussed different types of PCR. For more information on this topic visit different types of PCR.