Coupling of optimized multiplex PCR and automated capillary electrophoresis for efficient genome-wide searches

▼Genome-wide searches for linkage have come into frequent use to dissect complex genetic diseases and traits (Ref. 1, 2, 3, 4). The coelectrophoresis of dinucleotide repeat markers using multiple fluorescent labels has allowed investigators to efficiently reach the 10 cM resolution necessary to effectively screen the genome for linkage (Ref. 5, 6, 7). Despite the increased efficiency of coelectrophoresis, however, singleplex amplification of each marker remains a slow and cumbersome process. No one has described multiplex PCR conditions for an entire linkage mapping set. So, in order to increase throughput and decrease error rates, multiplex PCR was optimized for each of the three colors in each of the 28 panels of the ABI PRISM Linkage Mapping Set version 1(Perkin Elmer). These conditions should translate well for use with Linkage Mapping Set version 2, since 52% of the optimized multiplexes consist only of primer sets that were not replaced in the second version. Another 36% have only one primer set that was replaced, while the remaining 12% will probably not be useful. Figure 1 shows the typical progressions as primer concentrations within one multiplexed color are optimized. Amplification was carried out using 0.2 Units of AmpliTaq Gold DNA Polymerase (Perkin Elmer) in 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, and 250 μM of each dNTP for a total volume of 5 μL. Thermocycling was performed on a Perkin Elmer GeneAmp PCR System 9600(Perkin Elmer) using an initial hot start at 95 ◦C for 12 minutes, followed by 10 cycles of 94 ◦C for 15 seconds, 55 ◦C for 15 seconds, and 72 ◦C for 30 seconds, then 20 cycles of 89 ◦C for 15 seconds, 55 ◦C for 15 seconds, and 72 ◦C for 30 seconds, and a final extension step of 72 ◦C for 30 minutes. Primer concentrations were varied as shown above the position of each marker. Separation was carried out using capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The figure shows the electropherogram of only one of the four subjects used to optimize multiplex conditions. The multiplex shown is Panel 24 [TET], which proved quite difficult to optimize, finally requiring separation into two multiplexes. The bottom two electropherograms correspond to the multiplex of the two markers that failed to amplify within the larger multiplex. D17S928 overlapped with invariant nonspecific products of the multiplex, but this did not interfere with genotyping when all markers in the panel were pooled and electrophoresed. Two panels were run separately for singleplex and then multiplex PCR. The results are presented in (Table 1). Singleplex PCRs were optimized by modifying annealing temperatures and MgCl2 concentration. A comparison between the two methods shows a clear advantage for multiplex PCR in the ratio of marker standard deviation with average peak height, which corresponds to more even peak heights. The difference between the overall between markers standard deviation for the multiplexed markers, 530, and the between markers standard deviation within each multiplex PCR, 309, is the direct result of pooling four tubes of PCR products. By extrapolation, most of the higher standard deviation, relative to average peak height, for singleplex PCR