Optimization of semi-automated microsatellite multiplex polymerase chain reaction systems for rainbow trout ( Oncorhynchus mykiss)

Abstract

We have developed an octaplex and a hexaplex polymerase chain reaction (PCR) genotyping system for rainbow trout ( Oncorhynchus mykiss). During the development of the two multiplex systems, we encountered numerous allele scoring difficulties. The variable `plus A' modification (non-templated adenylation of the 3′ end of the amplified sequence by Taq polymerase) of PCR products across various DNA templates, in addition to the preferential amplification of smaller alleles over larger alleles (differential amplification) for certain loci, resulted in misidentified genotypes. The time spent correcting misidentified alleles greatly inhibited the efficiency of the semi-automated genotyping systems. We have determined that minimizing primer concentrations (ranging from 0.016 to 0.325 μM) and increasing Taq polymerase concentration (up to 3 units in a 15-μl sample volume) resulted in consistent `plus A' modification of alleles for all loci. Furthermore, increasing the KCl concentration from 50 to 100 mM during PCR amplification, alleviated the problem of differential amplification. These modifications virtually eliminated the need for manual editing of misidentified alleles for both multiplex PCR systems despite variation in template DNA quality and concentration. The information presented in this study will prove useful for those wishing to optimize a multiplex PCR system in the same or other species.