Two DNA fragments, where one or two of the primers incorporate the mutation site and the fragments have a 5'-terminal overlap, are amplified by 1stPCR.
The two amplified fragments are purified to remove the original primers and are mixed for a 2ndPCR amplification with the two flanking primers (primers A and D). Primers B and C are designed so that their 3'-regions correspond to the sequences adjacent to the deletion end points. The 1st PCRs with primers A + B and C + D will fail to copy the sequence defined for deletion. Either primer B or C must also contain a 5' extension sequence complementary to the other primer sequence.
The ends of the two PCR products will therefore be complementary and can be joined in the 2nd PCR. 2nd PCR reaction to yield a product that now lacks the region targeted for deletion.
