40 - Use of Polymerase Chain Reaction to Amplify Segments Outside Boundaries of Known Sequences

Abstract

The polymerase chain reaction (PCR) is an effective method for selectively amplifying specific DNA segments without resorting to conventional cloning procedures. Repeated cycles of synthesis result in a geometric increase in the number of copies of the region bound by the primers. The PCR does not allow the amplification of segments that lie outside the primers because an oligonucleotide that primes synthesis into a flanking region has no primer in the reverse direction and therefore produces only a linear increase in the number of copies. This chapter reviews the procedures adopted by several investigators to obtain fragments outside a region of known sequence and provide a protocol for one technique — inverse PCR. Many techniques for amplifying flanking regions of DNA are based on the creation of new primer-binding sites onto potential PCR templates either by enzymatic synthesis or by ligating oligonucleotides of known sequences to the ends of DNA fragments. For the amplification of complementary DNAs, it is usually sufficient to use a unique primer — one based on a region of previously determined sequence — along with a primer complementary to the enzymatically synthesized tail to generate a specific product.