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.