MegaPlex PCR: a strategy for multiplex amplification

Abstract

'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by coamplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.The key challenge in developing a high-multiplex amplification procedure is preventing excessive off-target priming by the many primers in the reaction [1][2][3] . Several previously developed methods use surface immobilization of primer pairs as a way to separate reactions and thus limit primerdimer formation [4][5][6] , but drawbacks of these methods include inefficient amplification, loss of reactants from the surface and considerable primer-dimer formation within pairs of primers.MegaPlex PCR likewise uses physical separation of primer pairs to prevent their interaction, but it additionally includes 'common sequences' at the 5′ ends of the surface primers so that early-stage amplicons can be moved into the solution phase for co-amplification by highly efficient and unbiased PCR using a single common primer pair [6][7][8] (Fig. 1). To reduce primer-dimer formation, MegaPlex PCR uses surface primers that are made partially double-stranded by base-pairing their common 5′ sequences with complementary 'barrier oligonucleotides'. A detailed methods description is available in Supplementary Methods online.During the development of MegaPlex PCR we evaluated a range of targets, input DNAs and reaction conditions. We used membrane arrays and microbeads as binding surfaces, with the beads now routinely attached to microtiter plate wells to provide a platform compatible with automation.Using the microbead support we found MegaPlex PCR to be effective with as little as 200 ng of human genomic DNA, and in various 15-plex reactions it recovered many different target sequences of up to at least 500 bp in size, with little bias against larger amplicons ( Supplementary Fig. 1 online). As occasional targets (~1/10 in preliminary studies) generated severe primer-dimer artifacts, regardless of the multiplex level of the experiment, optimization of MegaPlex PCR was required. One option was the exclusion of these targets by empirical or computational prefiltering, but we selected a modification that avoided the need for target preselection.This modification involves enriching the input genomic DNA for sequences of interest via a crude solution-phase multiplex PCR using specific primers for all the amplification targets. The primer sequences for this can match the specific portions of those on the solid surface, or they can be designed to prime slightly outside these sites in the genomic DNA. This strategy is used routinely in digital molecular counting and genome mapping studies that use limiting-dilution samples, with multiplexing up to at least 1,200 (ref. 9). Although this is less specific ...

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