使用READIT检测法分析凝血因子V莱顿突变。

Analysis of the factor V Leiden mutation using the READIT Assay.

作者信息

Rhodes R B, Lewis K, Shultz J, Huber S, Voelkerding K V, Leonard D G, Tsongalis G J, Kephart D D

机构信息

University of Minnesota, Minneapolis, MN, USA.

出版信息

Mol Diagn. 2001 Mar;6(1):55-61. doi: 10.1054/modi.2001.22327.

DOI:10.1054/modi.2001.22327

PMID:11257212

Abstract

BACKGROUND

A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation.

METHODS AND RESULTS

We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined.

CONCLUSION

Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.

摘要

背景

存在多种用于检测基因组DNA扩增片段中存在的单核苷酸多态性（SNP）的方法。我们展示了一种新型SNP评分工具在分析因子V莱顿突变中的应用。

方法与结果

我们开发了一种分析SNP的新方法。基于荧光素酶的技术，即READIT技术（普洛麦格公司，威斯康星州麦迪逊），用于分析从三个独立检测实验室送来进行因子V莱顿检测的510份剩余人类样本。对因子V莱顿突变进行了盲法回顾性分析，以确定该技术的准确性和通量能力。在READIT检测与检测实验室做出的基因型判定之间观察到100%的一致性。此外，在野生型和杂合子样本群体的均值之间观察到大于6个标准差的分离。对代表性野生型、杂合子和突变样本进行重复样本测量表明，杂合子和纯合子样本群体的均值之间有大于9个标准差的分离。确定了基于野生型、杂合子和突变样本群体均值的置信区间。

结论

使用READIT检测获得的完美一致性表明其作为一种SNP评分工具的有效性。因子V READIT检测的设计很简单，只需设计两个在倒数第二个3'位置不同的未修饰寡核苷酸，以与因子V序列的野生型或莱顿形式形成完美杂交体。使用先前发表的扩增引物和条件将优化和验证检测所需的时间减至最少。该检测提供的READIT计算器允许根据READIT检测数据进行自动基因型判定和统计分析。置信区间分析验证了使用READIT检测区分野生型、杂合子和突变样本的能力。