Extension Time In Pcr In King
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Extension Time Extensions are normally performed at 68°C. As a general rule, use extension times of one minute per 1000 base pairs (e.g. 3 minutes for a 3 kb product) For products less than 1 kb, use 45-60 seconds. Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions.
Duration of extension will be dependent upon amplicon size (30 sec per 1 kb). The period of elongation depends upon the desired length of the amplicon and the enzyme used. Since qPCR amplicons are short, this is typically 5–30 sec.
The final PCR step occurs at 70-75 ˚C and is known as extension. During this stage, DNA polymerase extends the DNA from the primers, creating new dsDNA with one old strand and one new strand.
During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times (typically 25-35 cycles), and because each new strand can also serve as a template for the primers, the region of interest is amplified exponentially.
DNA polymerase enzyme used in the PCR is obtained from bacteria that is found in thermal vents and hot springs. They thrive in such high temperatures and hence72∘C. is the optimum temperature for their enzymes to work at.
The extension time of PCR depends upon the synthesis rate of DNA polymerase and the length of target DNA. The typical extension time for Taq DNA Polymerase is 1 min/kb, whereas that of Pfu DNA polymerase is 2 min/kb.
A typical PCR cycle includes an extension step at 72°C after denaturation of double-stranded DNA and annealing of oligonucleotide primers. At this temperature the thermostable poly-merase replicates the DNA at an optimal rate that depends on the buffer and nature of the DNA template ( 1 ).
Extension: The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended.
Extension is achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double-stranded products of DNA. The temperature that is used during the extension phase is dependent on the DNA polymerase that is used.
