There are a lot of thermocycling and isothermal techniques for amplification of nucleic acids, such as Strand Displacement Amplification, Rolling Circle Amplification, Ligase Chain Reaction, Helicase Dependent DNA amplification, etc.
Thermocycling techniques of PCR
Thermocycling techniques use temperature cycling to drive repeated cycles of DNA synthesis.
Ligase Chain Reaction (LCR)
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 templates for subsequent cycles.
The main advantage of Ligase Chain Reaction is that a single point mutation in the original template DNA can prevent the reaction and an absence of product can be an indicator of mutations.
Isothermal techniques of PCR
Isothermal techniques do not rely on thermocycling.
They utilize 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.
Quantitative PCR (qPCR)
The amount of product that is synthesized during a set number of cycles of a PCR 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.
To carry out PCR where RNA is the starting material this method uses reverse transcriptase PCR, a process called RT–PCR (reverse transcriptase–PCR). 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.
Different types of PCR technique