Combining Multiplex and Touchdown PCR for Microsatellite Analysis

An improved nonradioactive polymerase chain reaction (PCR)-based method for simultaneous amplification of multiple loci of microsatellites has been developed as a rapid way to screen for microsatellites (1 ). The approach, termed multiplex-touchdown PCR (MT-PCR), is performed in a single PCR tube by combining touchdown (2 –5 ) and multiplex (6 ) PCR protocols. The touchdown format is used to improve the specificity and the quality of amplification, that is, only DNA bands of an expected size are present as the major PCR bands observed on the nondenaturing polyacrylamide gels, thereby overcoming the presence of differently sized background bands (a ladder-like problem). The multiplex strategy is used so that simultaneous amplification of multiple microsatellite loci is achieved. In this chapter, we describe the MT-PCR strategy that has been successfully used for simultaneous amplification of up to three mouse microsatellites by choosing primer pairs with the corresponding touchdown-PCR parameters. The MT-PCR is very useful for genotyping hybrid mice, provided the allelic size difference between two parental genotypes is amenable to separation by gel electrophoresis. In principle, this MT-PCR should be applicable to similar studies in other species, including humans.