Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue.
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.
Koizumi
Makoto
M
Lead Discovery Research Laboratories, Sankyo Company Ltd., 2-58, Hiromachi 1-Chome, Shinagawa-ku, Tokyo 140-8710, Japan. koizum@sankyo.co.jp
Morita
Koji
K
Takagi
Miho
M
Yasumo
Hiroaki
H
Kasuya
Atsushi
A
D000483:Alleles; D000091303:Angiopoietin-Like Protein 3; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005030:Ethylenes / Q000737:chemistry; D005838:Genotype; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011516:Prothrombin / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism
D000091303:Angiopoietin-Like Protein 3; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; C404495:Angptl3 protein, mouse; D017931:DNA Primers; D005030:Ethylenes; D036341:Intercellular Signaling Peptides and Proteins; D011516:Prothrombin; C036216:ethylene; D019914:Taq Polymerase