采用双色荧光偏振检测的入侵检测法对单核苷酸多态性进行基因分型。

Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection.

作者信息

Hsu T M, Law S M, Duan S, Neri B P, Kwok P Y

机构信息

Division of Dermatology, Washington University School of Medicine, St. Louis, MO 63110, USA.

出版信息

Clin Chem. 2001 Aug;47(8):1373-7.

PMID:11468224

Abstract

BACKGROUND

The PCR-Invader assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method.

METHODS

PCR product was incubated with Invader oligonucleotide and two primary probes at 93 degrees C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 degrees C for 5 min. FP measurements were made with a fluorescence plate reader.

RESULTS

Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average "no call" rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments.

CONCLUSIONS

The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.

摘要

背景

聚合酶链反应-入侵检测法（PCR-Invader 检测法）是一种可靠的均相检测法，已证明在单核苷酸多态性（SNP）标记基因分型中具有高度敏感性和特异性。在本研究中，我们引入了两项改进以优化该检测法：（a）通过在一个反应中检测每个 SNP 的两个等位基因来简化 PCR-入侵检测法；（b）采用荧光偏振（FP）作为检测方法来降低该方法的成本。

方法

将聚合酶链反应（PCR）产物与入侵寡核苷酸和两种一级探针在 93℃孵育 5 分钟。然后将与一级探针切割后的侧翼相对应的信号探针（用荧光素和 6-羧基四甲基罗丹明（TAMRA）染料标记）和切割酶 VIII（一种侧翼内切核酸酶）加入混合物中。该反应混合物在 63℃孵育 5 分钟。使用荧光酶标仪进行荧光偏振测量。

结果

以 PCR 产物为模板，对 88 名个体的一组 10 个 SNP 进行基因分型，共获得 880 个基因型。第一轮实验后观察到平均“无分型结果”率为 3.2%。对第一轮未能产生任何基因型的样本重新制备 PCR 产物，所有样本均得到明确的基因型。当比较 PCR-入侵检测法和采用 FP 的模板导向染料终止掺入检测法所确定的基因型时，所有 SNP 标记和实验的结果一致性达 100%。