A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

Abstract

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

“…Such primers have also been reported to increase the fluorescent signal of real-time PCR [56, 57]. In addition, inosine-flapped APLP primers improve the competitiveness of allele-specific primers to the template DNA, resulting in enhanced reliability of the SNP analysis [33]. The thermodynamics of the primers with inosine flaps has been proven to be less influenced by the sequence of PCR templates than the thermodynamics of the primers with 5′-flaps containing ordinary bases [33].…”

“…In addition, inosine-flapped APLP primers improve the competitiveness of allele-specific primers to the template DNA, resulting in enhanced reliability of the SNP analysis [33]. The thermodynamics of the primers with inosine flaps has been proven to be less influenced by the sequence of PCR templates than the thermodynamics of the primers with 5′-flaps containing ordinary bases [33]. Furthermore, we reported that our inosine-flapped APLP method in the multiplex SNP analysis showed markedly higher sensitivity than that reported in various studies [61].…”

“…PCR-APLP is an SNP typing method [25–33] based on PCR that uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method, at least two allele-specific primers and one “counter primer,” which serves as a common sense or antisense strand of the allele-specific primers, are required.…”

“…The allele-specific primers have SNP sites at the 3′-termini, and one of these primers should have a few noncomplementary flaps at the 5ʹ-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. We previously reported a more reliable SNP analysis method based on PCR-APLP using a 5ʹ-inosine flap primer, which improves the competitiveness of allele-specific primers [33]. In this study, we introduce a novel PCR-APLP sex determination method based on the detection of an SNP in exon 2 of the AMYL gene.…”

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

“…We recently developed novel APLP primers with a short inosine extension added to the 5′-terminus. This modification improves the competitiveness of allele-specific primers to the template DNA, resulting in enhanced reliability of the analysis of SNPs [ 28 ]. In the present study, we designed the primers based on this inosine-flapped APLP method.…”