I was asked about the concept and difference between PCR & sequencing and PCR-direct sequencing, I was confused about it. Is there someone who can help me? Thanks a lot!
One can sequence a PCR product in two ways: one is to clone the amplicon in a suitable vector and then it can be sequenced. This is followed when the PCR product is not pure enough! The other way is to directly sequence the PCR product after purification when you are sure that it is only a single amplicon present in the PCR. This is faster and easier compared to the cloning method.
I think direct sequencing refers to a method that allow the use of the PCR product directly in the sequencing reaction without going through the step of sample preparation (generally after the PCR you must perform a digestion step eg. with ExoSap).
One or both primers used for amplification can have a tail that allows the use of standard primers generally used for sequencing (eg. T 7 series).
One can sequence a PCR product in two ways: one is to clone the amplicon in a suitable vector and then it can be sequenced. This is followed when the PCR product is not pure enough! The other way is to directly sequence the PCR product after purification when you are sure that it is only a single amplicon present in the PCR. This is faster and easier compared to the cloning method.
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