A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays
A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays
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DOI：
10.1101/gr.10.7.1031
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发表时间：
2000-07-01
期刊：
影响因子：
7
通讯作者：
Syvänen, AC
中科院分区：
文献类型：
--
作者：
Pastinen, T;Raitio, M;Syvänen, AC
这项研究描述了一个实用的系统，它允许高通量的单核苷酸多态(SNPs)基因分型和通过引物阵列上的等位基因特异性延伸检测突变。该方法依赖于在优化的反应条件下，通过逆转录酶(RT)酶对两个定义等位基因的3‘-核苷酸不同的固定化等位基因特异引物的序列特异性延伸。我们通过为40个突变或SNPs产生8000多个基因类型，展示了在低冗余的斑点引物阵列上执行这一简单的一步程序的潜力。这些基因类型形成了三个易于识别的聚类群，所有已知的基因类型都被正确分配。由于在单个反应中存在100多个扩增片段时，不同基因型之间的鉴别力保持不变，因此该系统将有可能实现更高程度的多重反应。酶辅助反应提供了高度特异性的等位基因区分，其能力证明了它能够检测存在于5%的样本中的多个位置的少数序列变异。该分析格式基于微型反应室，在标准384孔间距的显微镜载玻片上携带阵列，每个SNP用于80个样品，导致试剂消耗低，并使大量样品的平行分析变得方便。在该实验中，使用一个或两个荧光核苷酸类似物作为标记，因此可以用现有的阵列扫描仪和软件来解释基因分型结果。这里描述的基于阵列的基因分型系统的一般可访问性、简单的设置和稳健的程序将提供一种简单的方法来增加任何分子生物学实验室的SNP分型的吞吐量。
This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.