Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays
Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays
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DOI：
10.1101/gr.7.6.606
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发表时间：
1997-06-01
期刊：
影响因子：
7
通讯作者：
Syvanen, AC
中科院分区：
文献类型：
--
作者：
Pastinen, T;Kurg, A;Syvanen, AC
我们描述了一种用于多重检测突变的方法，其中将固相微测序原理应用于寡核苷酸阵列格式。通过使用DNA聚合酶延伸固定化引物来检测突变，所述固定化引物与突变核苷酸位置紧邻的模板序列退火，所述模板序列具有单个标记的双脱氧核苷三磷酸。通过在小的玻璃区域上偶联每个待检测突变的一个引物来制备阵列。在多重PCR反应中扩增跨越九个疾病突变的基因组片段（其被选择作为测定的靶标），并用作引物阵列上的微型测序反应的模板。通过高度特异性引物延伸反应，在每个分析的核苷酸位置明确定义纯合基因组DNA样品的基因型。在与固定化等位基因特异性探针在相同的测定格式的杂交相比，纯合和杂合基因型之间的区分能力是一个数量级更高的使用minisequencing方法。因此，单核苷酸引物延伸是一个有前途的原则，为未来的高通量突变检测和基因分型，使用高密度DNA芯片技术。
We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area, Genomic fragments spanning nine disease mutations, which were selected as targets For the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.