title: 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.
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi, 409-3898, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shojo
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi, 409-3898, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi, 409-3898, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Takahashi
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi, 409-3898, Japan.
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Kakuda
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi, 409-3898, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Adachi
mesh_terms: D000483:Alleles; D017931:DNA Primers; D006801:Humans; D007288:Inosine / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D017931:DNA Primers; D007288:Inosine