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.