Polymerase chain reaction was developed in 1983 by Kary Mullis. To date, there are many different types of PCR technique.
Some of them are RT-PCR, touchdown PCR, real time PCR, nested PCR, Strand Displacement Amplification, Rolling Circle Amplification, Ligase Chain Reaction, Helicase Dependent DNA amplification, etc.
2 groups of different types of polymerase chain reaction are thermocycling PCR techniques and isothermal amplification methods.
Different types of PCR technique based on thermocycling (heating and cooling steps)
Thermocycling techniques use temperature cycling to drive repeated cycles of DNA synthesis.
Multiplex PCR is a type of PCR technique which allows an amplification of many target sequences concurrently in the same reaction mixture.
A single reaction mixture includes sets of primer pairs for different DNA targets. It reduces the consumption of PCR reagents, and, at the same time, imposes restrictions on used primers.
To work properly within one reaction, sets of primers must be optimized. They have to have similar annealing temperatures and produce amplicons of different sizes to form distinct gel electrophoresis bands for the followed PCR analysis.
Nested PCR is used to increase the specificity of a DNA amplification reducing unspecific products.
This technique utilizes two sets of primers.
The first set allows a first polymerase chain reaction. The product of this reaction serves as a source of target DNA to a second PCR using the second set of primers.
This type of polymerase chain reaction serves to reduce non-specific amplification during the initial set up stages.
Hot-start PCR technique keeps the DNA polymerase in an inactive state at temperatures lower than an annealing temperature.
This modification prevents the amplification during reaction setup when primers bind to DNA sequences with low homology.
Two variants of this technique are mechanical and non-mechanical hot start PCR.
Mechanical hot start PCR performed by heating the reaction mixture to the DNA melting temperature before adding the Taq polymerase.
Non-mechanical hot start PCR uses specialized enzyme systems which inhibit an activation of the DNA polymerase at room temperature.
Touchdown PCR is another technique to reduce nonspecific amplification.
It is achieved by raising the annealing temperature above the melting temperature of the used primers in the initial cycles and lowering in the later cycles.
The higher temperatures during the initial cycles help primers to bind to DNA templates with greater specificity while the lower temperatures allow more efficient amplification from the produced amplicons.
Ligase Chain Reaction (LCR)
This type of PCR technique uses four primers for DNA amplification (two primers for each strand of the DNA target).
Ligase Chain Reaction primers are much longer than usual PCR primers and designed to cover the entire sequence to be amplified.
During the first annealing step, primers are sealed by a thermostable DNA ligase.
This generates a fragment that is as long as the total length of each pair of primers which serves as the DNA templates for subsequent cycles.
The main advantage of Ligase Chain Reaction is that a single point mutation near the junction in the original template DNA can prevent the reaction and an absence of product can be an indicator of mutations.
Quantitative PCR (qPCR)
The amount of product that is synthesized during a set number of cycles of a polymerase chain reaction depends on the number of DNA molecules that are present in the starting mixture. This enables PCR to be used to quantify the number of DNA molecules present in an extract.
In quantitative PCR the amount of product synthesized during a test PCR is compared with the amounts synthesized during PCRs with known quantities of starting DNA.
Today, quantification is carried out by real-time PCR - a modification of the standard PCR technique in which synthesis of the product is measured over time.
More frequently this method is used to measure RNA amounts.
For example to determine the expression of a particular gene in cancerous cells. This method allows monitoring the development of cancer.
Reverse transcription PCR
To carry out polymerase chain reaction where RNA is the starting material this method uses reverse transcriptase, a process called RT–PCR (reverse transcriptase polymerase chain reaction).
The first step in this method is to convert the RNA molecules into single-stranded complementary DNA (cDNA). After this step, the experiment proceeds as in the standard technique.
Some thermostable polymerases, such as Tth, have a reverse transcriptase activity under certain buffer conditions and able to make DNA copies of both RNA and DNA molecules.
TaqMan PCR is one of the real-time PCR techniques.
It uses an oligonucleotide probe which is complementary to an internal sequence within the amplified strands.
It has a fluorescent group at one end and a quencher at another end. As long as both fluorophore and quencher stay within the oligonucleotide probe, no fluorescence is emitted.
During DNA amplification, the oligonucleotide probe, and the primers will bind to newly synthesized strands. The polymerase will destroy the probe due to the intrinsic 5′→3′ exonuclease activity and release the fluorophore.
The intensity of the fluorescence is proportional to the amount of generated product.
Assembly PCR or Polymerase Cycling Assembly was developed to produce novel long nucleic acid sequences.
The main difference from traditional polymerase chain reaction is the length and quantity of primers.
To synthesize artificial oligonucleotide, assembly PCR is performed on long, up to 50 nucleotides, primers. These primers have short overlapping segments and alternate between sense and antisense directions covering the entire target sequence.
During successive cycles, the primers hybridize by complementary segments and then polymerase increases the length of fragments producing the final long nucleic acid sequence.
Typically, the assembly phase is followed by a regular polymerase chain reaction with end primers to increase the amount of the final product.
Different types of PCR technique based on isothermal amplification of DNA
Isothermal techniques do not rely on thermocycling. They are easier to operate and require less energy than standard PCR methods.
These types of PCR utilize DNA polymerases with strand-displacement activity.
Loop-mediated isothermal amplification (LAMP)
PCR reaction mixture for loop-mediated isothermal amplification has strand displacement-type DNA synthetase instead of Taq polymerase. The mixture is held at a constant temperature (about 65 °C) to promote the reaction.
Rolling Circle Amplification (RCA)
With Rolling Circle Amplification, first two ends of the DNA of interest are joined together using a DNA ligase to form a circular single-stranded DNA template. The primer is attached to this template during an annealed step. The amplification is carried out to complete the circle. The synthesized strand is displaced due to the property of the polymerase with strand-displacement activity.
The main advantage is that the synthesis can occur at room temperature.
Helicase-Dependent DNA Amplification (HDA)
Helicase-Dependent DNA Amplification relies on a DNA helicase to separate the double-stranded DNA.
The major advantage is that the helicase can operate at room temperature.
Different types of polymerase chain reaction technique
References & further readings
- Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19(14):4008.
- Haff LA. Improved quantitative PCR using nested primers. PCR Methods Appl. 1994;3(6):332-7.
- Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci USA. 1999;96(16):9236-41.
- Assembly PCR for novel gene synthesis