A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples.
A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples.
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DOI：
10.1016/j.legalmed.2004.05.005
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发表时间：
2004-10-01
期刊：
影响因子：
--
通讯作者：
Ishizu, Hideo
中科院分区：
文献类型：
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作者：
Doi, Yusuke;Yamamoto, Yuji;Ishizu, Hideo
我们开发了一种新的ABO基因分型方法，使用多重单碱基引物延伸反应。该方法允许同时检测ABO基因中的6个SNP位点（nt 261、297、681、703、802和803），并从其组合中确定ABO基因型。它能够对从103名日本人中提取的外周血DNA的所有样品进行ABO基因分型，并且具有高度令人满意的检测灵敏度，能够对0.1ng基因组DNA进行基因分型。用此方法可对微量血痕、加热血痕、老化血痕及混合血痕进行ABO基因型鉴定。对26种动物样本和细菌样本进行的实验表明，该方法的物种特异性表明，黑猩猩和大猩猩的基因分型是可能的，但它们的基因型在人类中极为罕见。此外，我们将这种方法应用于个案样本，并成功地确定了ABO基因型的骨骼，牙齿，肌肉，器官，指甲，精液污染的阴道液中，ABO分组通过传统的血清学技术是不可能的。这种新方法能够通过两种特异性反应，即PCR和引物延伸反应，灵敏地同时检测ABO基因中的六个SNP位点。因此，它有望成为一种有效的ABO基因分型方法，特别是法医样本。
We developed a new method for ABO genotyping using a multiplex single-base primer extension reaction. The method allows for the simultaneous detection of six SNP sites in the ABO gene (nt 261, 297, 681, 703, 802, and 803) and the determination of ABO genotypes from their combinations. It enabled ABO genotyping of all samples of peripheral blood DNA extracted from 103 Japanese individuals, and had a highly satisfactory detection sensitivity being capable of genotyping 0.1 ng of genomic DNA. Using this method, we were able to determine ABO genotypes of minute stain samples, heated bloodstains, aged bloodstains and mixed samples. Experiments with samples from 26 animal species and bacterial samples to test the species-specificity of the method showed that genotyping was possible in the chimpanzee and gorilla, but their genotypes were extremely rare in humans. In addition, we applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal fluid in which ABO grouping by conventional serological techniques was not possible. This new method enables the sensitive, simultaneous detection of six SNP sites in the ABO gene by two specific reactions, i.e. PCR and a primer extension reaction. Therefore, it holds promise as an effective method of ABO genotyping particularly for forensic samples.