Molecular Techniques and Methods

Deletion Mutagenesis by Overlap Extension and Splicing PCR

Copy Right © 2001/ Institute of Molecular Development LLC

  • 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 1st PCR.
  • The two amplified fragments are purified to remove the original primers and are mixed for a 2nd PCR 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.

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