Mung bean nuclease treatment improves Se genotyping by allele-specific inverse polymerase chain reaction...

The allele-specific inverse polymerase chain reaction (PCR) technique, which has been explored to detect two linked polymorphic regions simultaneously, was applied to genotype the Se system. The major alleles of the Se system in Japanese are Se, sej defined by a single nucleotide substitution in the Se allele, and se(fus) generated by recombination between the Sec1 and FUT2 genes. The first PCR products using gene-specific primers were self-ligated, and each allele was detected by the second inverse-PCR using allele-specific primers. The 340, 331 and 353-bp products were finally amplified from Se, sej and se(fus) templates, respectively. The first PCR products without mung bean nuclease treatment were not self-ligated and non-specific fragments were amplified in the second PCR, suggesting that non-templated adenylation occurred at the termini during the first PCR. Nuclease digestion of the first PCR products that blunts their termini was found to reduce interference of non-templated adenylation with the intramolecular ligation and to improve the genotyping markedly. This modified allele-specific inverse-PCR method is applicable to analyze haplotypes consisting of separated single nucleotide polymorphisms and recombinant genes.