Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G) |
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Authors: | Ginya Harumi Asahina Junko Yoshida Mamiko Segawa Osamu Asano Tsutomu Ikeda Hideo Hatano Yukiko Miyashita Shishido Madoka Johansson Britt-Marie Zhou Qin Hallberg Mathias Takahashi Masaaki Nyberg Fred Tajima Hideji Yohda Masafumi |
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Affiliation: | Precision System Science, Kamihongou, Matsudo, Chiba 271-0064, Japan. harumi.ginya@pss.co.jp |
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Abstract: | We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals. |
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Keywords: | SNP OPRM1 CYP2C19 Allele-specific oligonucleotides Competitive hybridization |
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