Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation.
Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation.
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多重荧光固相小测序可有效筛选 DNA 序列变异。
DOI：
10.1093/clinchem/42.9.1391
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发表时间：
1996
影响因子：
9.3
通讯作者：
A. Syvänen
中科院分区：
文献类型：
--
作者：
T. Pastinen;J. Partanen;A. Syvänen
我们开发了一种多重固相微测序方法来检测未分裂样品中的多个单核苷酸多态性。扩增的DNA模板首先在歧管上捕获。然后，使用多个不同大小的微序列引物，与荧光标记的双脱氧核苷酸同时进行单核苷酸延伸反应。扩展产物的大小，通过使用DNA测序仪器确定，定义了多态性的位点，并且结合的核苷酸给出了每个位点上核苷酸的身份。以HLA-DQA1分型为模型系统对该方法进行评价。将HLA-DRB1基因的DR2亚群与DQA1基因一起分型，以证明该方法在同时分析多个位点的多个基因时的可行性。该方法通常适用于筛选任何单核苷酸多态性或点突变，其流形格式允许大量样品的实际处理。
We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.