25 - Direct Analysis of Specific Bonds from Arbitrarily Primed PCR Reactions

Abstract

This chapter describes two variations on the arbitrarily primed PCR (AP-PCR) technique that allow the easy analysis of AP-PCR products. The first, sequencing with arbitrary primer pairs, uses two AP-PCR primers in a single AP-PCR reaction. One of these can then be used as a sequencing primer for direct double-stranded sequencing of the DNA band. In the second technique, constant plus arbitrary primer amplification, one primer is held uniform in every AP-PCR reaction and is paired with a variety of different arbitrary primers. The constant primer may then be modified, thereby allowing the PCR product to be modified in specific ways. Sequence variation can be analyzed by denaturing gradient gel electrophoresis (DGGE). This variation may also be screened for by fluorescent single-strand polymorphism analysis (SSCP), in which the constant primer is attached to a fluorescent label, and the product analyzed on an SSCP gel in the ABI automated sequencing apparatus using Genescan software. The AP-PCR for specific band analysis requires three stages for the generation of specific usable bands. 1) AP-PCR using primers singly and in combination. Running these reactions in parallel on agarose gels ensures that the bands to be analyzed are the product of the two different PCR primers and are not produced by either primer alone. 2) Agarose gel electrophoresis for identification and excision of appropriate bands. 3) Reamplification of DNA in the excised band using the same primer pair with or without specific modifications to the constant primer.