Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue

• Published in: Analytical biochemistry Vol. 340; no. 2; pp. 287 - 294
• Main Authors: Koizumi, Makoto, Morita, Koji, Takagi, Miho, Yasumo, Hiroaki, Kasuya, Atsushi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-05-2005
• Subjects: Allele-specific polymerase chain reaction; Alleles; Angiopoietin-like Proteins; Angiopoietins; Animals; AS–PCR; Base Sequence; BNA; DNA Primers - chemistry; ENA; Ethylenes - chemistry; Genotype; Humans; Intercellular Signaling Peptides and Proteins - genetics; LNA; Mice; Modified nucleoside; Polymerase Chain Reaction - methods; Polymorphism, Single Nucleotide - genetics; Prothrombin - genetics; SNP; Taq Polymerase - metabolism; BNA; SNP; ENA; Allele-specific polymerase chain reaction; AS–PCR; Modified nucleoside; LNA
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.02.029

When we placed an ENA residue into primers at the 3′ end, or the
n-1,
n-2, or
n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3′ end, only primers containing the ENA residue at the
n-2 position were read by
Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer–dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS–PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by
Taq DNA polymerase in the modified primer–template duplex. These results demonstrate that ENA primer-based AS–PCR would enable a rapid and reliable technique for SNP genotyping.