A novel SNP detection technique utilizing a multiple primer extension (MPEX) on a phospholipid polymer-coated surface.
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.
Imai
Kazuhide
K
Laboratory of Medical Genomics, Department of Human Genome Technology, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, Japan.
Ogai
Yasukazu
Y
Nishizawa
Daisuke
D
Kasai
Shinya
S
Ikeda
Kazutaka
K
Koga
Hisashi
H
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009061:Mouth Mucosa / Q000166:cytology; D015345:Oligonucleotide Probes; D010743:Phospholipids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers; D020641:Polymorphism, Single Nucleotide; D012016:Reference Values; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D015345:Oligonucleotide Probes; D010743:Phospholipids; D011108:Polymers; D004247:DNA