![]() ![]() 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. 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. 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. Ligase Chain Reaction primers are much longer than usual PCR primers and designed to cover the entire sequence to be amplified.ĭuring the first annealing step, primers are sealed by a thermostable DNA ligase. This type of PCR technique uses four primers for DNA amplification (two primers for each strand of the DNA target). 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. 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. Touchdown PCR is another technique to reduce nonspecific amplification. Non-mechanical hot start PCR uses specialized enzyme systems which inhibit an activation of the DNA polymerase at room temperature. Mechanical hot start PCR performed by heating the reaction mixture to the DNA melting temperature before adding the Taq polymerase. Two variants of this technique are mechanical and non-mechanical hot start PCR. This modification prevents the amplification during reaction setup when primers bind to DNA sequences with low homology. ![]() Hot-start PCR technique keeps the DNA polymerase in an inactive state at temperatures lower than an annealing temperature. This type of polymerase chain reaction serves to reduce non-specific amplification during the initial set up stages. The product of this reaction serves as a source of target DNA to a second PCR using the second set of primers. The first set allows a first polymerase chain reaction. This technique utilizes two sets of primers. Nested PCR is used to increase the specificity of a DNA amplification reducing unspecific products. They have to have similar annealing temperatures and produce amplicons of different sizes to form distinct gel electrophoresis bands for the followed PCR analysis. To work properly within one reaction, sets of primers must be optimized. It reduces the consumption of PCR reagents, and, at the same time, imposes restrictions on used primers. Multiplex PCR is a type of PCR technique which allows an amplification of many target sequences concurrently in the same reaction mixture.Ī single reaction mixture includes sets of primer pairs for different DNA targets. ![]() Thermocycling techniques use temperature cycling to drive repeated cycles of DNA synthesis. Different types of PCR technique based on thermocycling (heating and cooling steps) ![]()
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