A simple method to score single nucleotide polymorphisms based on allele-specific PCR and primer-induced fragment-length variation

We describe a simple protocol to genotype single nucleotide polymorphisms (SNPs), which combines allele-specific polymerase chain reaction (PCR) with fragment-length analysis. Three primers are used in the PCR: two allele-specific forward primers with a length-difference and one reverse primer. The forward primers induce a length-difference between the SNP-variants, which can be assessed with standard fragment-length analyses. We designed primers for 21 SNPs, and codominance was achieved for 76% of these SNPs. An inexpensive and flexible laser-detection scoring protocol can be achieved with multiplex scoring and by incorporating the M13(-21) genotyping method.