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
Precision System Science, Kamihongou, Matsudo, Chiba 271-0064, Japan.
Anal Biochem. 2007 Aug 1;367(1):79-86. doi: 10.1016/j.ab.2007.04.052. Epub 2007 May 6.
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.
我们之前开发了一种三维微阵列系统——生物链（Bio-Strand），它与我们的磁珠分离技术（Magtration Technology）相结合，在自动化DNA分析方面展现出优势。在当前研究中，我们为生物链开发了一种紧凑型系统——便捷生物链（Handy Bio-Strand），它由几种用于制备生物链尖端、杂交和检测的工具组成。使用便捷生物链，我们通过等位基因特异性寡核苷酸竞争性杂交（ASOCH）对OPRM1（A118G）进行了单核苷酸多态性（SNP）基因分型。含有SNP位点的DNA片段通过聚合酶链反应（PCR）从基因组DNA中扩增出来，然后固定在微孔尼龙线上。这样，制备好的生物链尖端与等位基因特异性的Cy5探针（<15mer）杂交，SNP位点设计位于探针中心。通过优化竞争剂的用量，Cy5探针的选择性提高，而信号没有大幅下降。使用便捷生物链通过ASOCH对从全血样本制备的23个人类基因组的OPRM1（A118G）基因型进行了测定。结果与通过PCR直接测序测定的结果完全一致。使用便捷生物链进行ASOCH对于小型实验室和医院的SNP基因分型将是一种非常简单且可靠的方法。
