Nested Patch PCR is a method designed to identify SNPs and mutations across many targeted loci for many samples in parallel. In the July issue of Cold Spring Harbor Protocols, Robi Mitra and colleagues from Washington University present Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci, a method where a large number (greater than 90) of targeted loci from genomic DNA are simultaneously amplified in the same reaction. These amplified loci can then be sequenced on a second-generation sequencing machine to detect single nucleotide polymorphisms (SNPs) and mutations.

Methods that employ mulitplexing during PCR reactions are often hampered by increased interprimer interactions that inhibit uniform amplification and increased formation of mispriming products. The protocol presented here was designed to reduce these two problems and results in a high specificity, with 90% of sequencing reads mapping to targeted loci. Nested Patch PCR is well-suited for the amplification of an intermediate number (100-1000) of targeted regions across a large number of samples and it offers a simple workflow that is compatible with 96-well plates and sample-specific DNA barcodes.