Abstract:
: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 micro opioid receptor gene. We immobilized 20 allele-specific oligonucleotides on this substrate, and substantially reproduced the results previously obtained by other methods.
journal_name
Mol Biosystjournal_title
Molecular bioSystemsauthors
Imai K,Ogai Y,Nishizawa D,Kasai S,Ikeda K,Koga Hdoi
10.1039/b701645jsubject
Has Abstractpub_date
2007-08-01 00:00:00pages
547-53issue
8eissn
1742-206Xissn
1742-2051journal_volume
3pub_type
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