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Research at the interface between chemistry and the -omic sciences and systems biology.
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Mol. BioSyst., 2007, 3, 547 - 553, DOI: 10.1039/b701645j
A novel SNP detection technique utilizing a multiple primer extension (MPEX) on a phospholipid polymer-coated surface
Kazuhide Imai, Yasukazu Ogai, Daisuke Nishizawa, Shinya Kasai, Kazutaka Ikeda and Hisashi Koga
Conventional methods for detecting single nucleotide polymorphisms (SNPs), including direct DNA sequencing, pyrosequencing, and melting curve analysis, are to a great extent limited by their requirement for particular detection instruments. To overcome this limitation, we established a novel SNP detection technique utilizing multiple primer extension (MPEX) on a phospholipid polymer-coated surface. This technique is based on the development of a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers extraordinarily stable thermal properties, as well as chemical properties advantageous for enzymatic reactions on the surface. To visualize allele-specific PCR products on the surface, biotin-dUTP was incorporated into newly synthesized PCR products during the extension reaction. The products were ultimately detected by carrying out a colorimetric reaction with substrate solution containing 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). We demonstrated the significance of this novel SNP detection technique by analyzing representative SNPs on 4 LD blocks of the µ opioid receptor gene. We immobilized 20 allele-specific oligonucleotides on this substrate, and substantially reproduced the results previously obtained by other methods.
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