SNP-SCALE: SNP scoring by colour and length exclusion

Here we present SNP-SCALE, a new single nucleotide polymorphism genotyping protocol based on allele-specific polymerase chain reaction (AS-PCR), but with two critical modifications designed to maximize accuracy, efficiency and throughput and minimize costs. The first is the addition of a locked nucleic acid (LNA) at the 3'-SNP position of each of the two allele-specific forward primers. The second is the addition of size-differentiating M13 tails at the 5' end of each primer, which facilitates the use of universal fluorescent primers. The use of LNAs makes SNP-SCALE significantly more sensitive and specific than standard AS-PCR. Moreover, SNP-SCALE is ideally suited to a touchdown-like PCR protocol, such that PCRs can be standardized, PCR optimization is straightforward, and medium-high throughput can be readily achieved. We anticipate that the SNP-SCALE protocol will be useful to molecular ecologists for a broad range of studies.