Polymerase chain reaction annealing temperature

Figure 3.25 Polymerase chain reaction. The steps involved in the chain reaction are as follows (i) Incubation of the DNA at a temperature above 90 °C in order to separate the two strands of the DNA duplex, (ii) Cooling of the solution to about 50 °C to allow annealing of the primers to the template (i.e. the nucleotides bind to the template DNA according to the basepairing rules), (iii) Finally, addition of the polymerase and Mg ions to extend the nucleotide primer and complete the synthesis of the complementary DNA, which takes place at about 70 °C. (iv) The sequence (i) to (iii) is repeated to allow another extension to occur many repetitions can be carried out which results in enormous multiplication of the DNA strands. NTPs - deoxyri-bonucleoside triphosphates.

One method of amplifying DNA in a polymerase chain reaction is through temperature cycling. The double-stranded DNA is denatured between 90°C and 100°C, while the annealing and extension demands lower temperatures. Sufficient amplification of the DNA occurs only after many cycles of temperature change. 

Immunocapture-polymerase chain reaction (IC-PCR) is a synthesis of two commonly used diagnostic tools. This method exploits the high-affinity binding of antibodies to provide a facile method of purification, usually from a complex matrix, supplying the substrate for PCR detection. PCR exponentially amplifies a deoxyribonucleic acid (DNA) template in a temperature-dependent fashion by the annealing of oligonucleotide primers, enzymatic extension of bound primers by a heat-stable polymerase, followed by denaturation of... 

The polymerase chain reaction. Double-stranded ONA is heated to 9S"C in (he presettce of two short oligonucleotide primer sequences, each of which is complementary to the end of one of the strands. After the DNA denatures, the temperature is lowered and the primer sequences anneal to the strand ends. Raising the temperature in the presence of Tag polymerase, Mg, and a mixture of the four deoxynucleotide triphosphates (cINTP s) effects strand replication, producing two DNA copies. Each further repetition of the sequence again doubles the number of copies. 

The polymerase chain reaction depends on repeated cycles of separation of DNA strands followed by annealing of primers. The first step requires a significantly higher temperature than the second, giving rise to the requirement for strict temperature control. 

The melting temperature (T ) of nucleic acids defines the thermal point at which 50% of the complementary molecule dissociate/melt away from their cognate strands or anneal/hybridize the two strands. The capacity to estimate melting temperatures is important because it helps the researcher select strand separation/hybridization temperatures, such as in polymerase chain reaction and microarray assays. A good approximation of melting tonperature for oligonucleotides takes the expression (Wallance et al., 1979) ... 

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