Nested PCR

[ishla]

Nested PCR

• Quote

Post by ishla » Mon Jul 07, 2008 10:38 am

Can you use one of the primers you have used in the first round of your nested PCR in the second round along with a new primer situated within the first round product???

[Cat]

• Quote

Post by Cat » Mon Jul 07, 2008 1:37 pm

Yes you can, but expect artifacts along with your expected product.

[dipjyoti]

• Quote

Post by dipjyoti » Tue Jul 08, 2008 9:34 am

Nested PCR means that two pairs of PCR primers were used for a single locus.
The selection of alternative and similar primer binding sites gives a selection of products, only one containing the intended sequence. The product from the first reaction undergoes a second run with the second set of primers.
It is very unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product from the second PCR has little contamination from unwanted products of primer mis-binding and alternative primer target sequences.

[Cat]

• Quote

Post by Cat » Wed Jul 09, 2008 2:50 pm

Ishla is actually doing semi-nested PCR: one primer is the same in both rounds. That means that single primer amplification artifacts are possible.