Under optimal conditions, DNA polymerase will add about 1,000 bp/minute. In this step, the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.Īlso known at extension, this step usually occurs at 72-80☌ (most commonly 72☌). The reaction temperature is rapidly lowered to 54-60☌ for 20-40 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds. This step involves heating the reaction mixture to 94☌ for 15-30 seconds. Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation also important for polymerase activity, stability and fidelity.Īll the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine.Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70☌.DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.The PCR reaction requires the following components: DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double stranded DNA. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. The PCR involves the primer mediated enzymatic amplification of DNA.
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