Reliable and Fast Allele-Specific Extension of 3′-LNA Modified Oligonucleotides Covalently Immobilized on a Plastic Base, Combined with Biotin-dUTP Mediated Optical Detection
Reliable and Fast Allele-Specific Extension of 3′-LNA Modified Oligonucleotides Covalently Immobilized on a Plastic Base, Combined with Biotin-dUTP Mediated Optical Detection
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共价固定在塑料基底上的 3-LNA 修饰寡核苷酸的可靠且快速的等位基因特异性延伸，结合生物素-dUTP 介导的光学检测
DOI：
10.2116/analsci.22.1537
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发表时间：
2006
影响因子：
1.6
通讯作者：
T. Imai
中科院分区：
文献类型：
--
作者：
Y. Michikawa;K. Fujimoto;K. Kinoshita;Seiko Kawai;K. Sugahara;T. Suga;Yoshimi Otsuka;K. Fujiwara;M. Iwakawa;T. Imai
在目前的工作中，一个方便的微阵列SNP分型系统已开发使用塑料基地，共价固定氨基修饰的寡核苷酸。通过对固定化寡核苷酸引物进行等位基因特异性增强的酶促延伸，并在SNP识别3′端核苷酸处进行锁核酸（LNA）修饰，实现了可靠的SNP等位基因识别。在引物延伸过程中掺入多个生物素-dUTP分子，然后结合碱性磷酸酶偶联的链霉亲和素，通过将有色碱性磷酸酶底物沉淀到塑料基底表面上，允许对基因分型结果进行光学检测。值得注意的是，在没有双链模板DNA的初步退火步骤的情况下证明了快速引物延伸，允许在几个小时内进行整个过程。同时评估基因TGFB 1，SOD 2和APEX 1中的三个SNP，以前研究与辐射敏感性的关联，在25个人中显示出完美的分配与另一种已建立的技术（MassARRAY系统）获得的数据。
In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3′-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).