Extension Time In Pcr In Sacramento
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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.
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 ).
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.
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.
Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low. If the extension time is too short, there will be insufficient time for complete replication of the target. Generally, use an extension time of 1 min/kb.
Extension: The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended.
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.
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.
Using too few PCR cycles can lead to insufficient amplification. Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low. If the extension time is too short, there will be insufficient time for complete replication of the target.
Increasing the final extension time improves full-length replication and yield of a 0.7-kb, GC-rich PCR fragment from human gDNA in these experiments. The smear under the desired band in 0 minute final extension suggests incomplete extension of the PCR amplicon by the DNA polymerase.
