I am trying to measure telomere length in human DNA samples through qPCR. In the protocol, they have not included extension step. Can anybody explain how a qPCR can work without an extension step? Telomere is nothing but a repeated sequence of TTAGGG.
Dear Dr. Sowmya N Rao
In qPCR, the annealing/extension can be a one step process instead of two steps because most of the amplicons are typically short. Primer extension is most efficient at 72°C because this is the optimal working temperature for most DNA polymerases. However, polymerases have sufficient activity even at lower temperatures (at 60°C) that is sufficient to amplify shorter templates.
Since qPCR amplicons are typically shorter ( 75-150 bp in length) than conventional PCR products, you may combine the extension with annealing in a single step. This will be much quicker than the three-step process as less time will be taken as well as it will help gain specificity.
Best.
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