Multiplex automated primer extension analysis: Simultaneous genotyping of several polymorphisms

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is of significant scientific importance for linkage and association studies. We report here an automated fluorescent method we call multiplex automated primer extension analysis (MAPA) that can accurately genotype multiple known SNPs simultaneously. This is achieved by substantially improving a commercially available protocol (SNaPshot (TM)). This protocol relies on the extension of a primer that ends one nucleotide 5' of a given SNP with fluorescent dideoxy-NTPs (minise-quencing), followed by analysis on an ABI PRISM (R) 377 Semi-Automated DNA Sequencer. Our modification works by multiplexing the initial reaction that produces the DNA template for primer extension and/or multiplexing several primers (corresponding to several SNPs) in the same primer extension reaction. Then, we run each multiplexed reaction on a single gel lane. We demonstrate that MAPA can be used to genotype up to four SNPs simultaneously, even in compound heterozygote samples, with complete accuracy (based on concordance with sequencing results). We also show that primer design, unlike the DNA template purification method, can significantly affect genotyping accuracy, and we suggest useful guidelines for quick optimization.