Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests.
Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests.
复制标题
基于变性梯度的二维电泳基因突变扫描测试的快速设计。
DOI：
10.1093/nar/26.10.2398
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发表时间：
1998
影响因子：
14.9
通讯作者：
Vijg,J
中科院分区：
文献类型：
--
作者：
vanOrsouw,NJ;Dhanda,RK;Rines,RD;Smith,WM;Sigalas,I;Eng,C;Vijg,J
随着目前人类疾病基因的快速鉴定，需要实用的、具有成本效益的基因筛选测试。基于大小和碱基对序列，分别在非变性和变性梯度聚丙烯酰胺凝胶中对PCR扩增的基因片段进行二维电泳分离，为基因突变扫描提供了一种快速的平行方法。该系统的变性梯度凝胶电泳（DGGE）组件的准确性在很大程度上取决于PCR引物的设计和它们所包含片段的熔解特性。我们已经开发出一个完全自动化的普遍适用的程序，以最少的时间和精力生成最佳的二维测试设计。设计的RB1，TP53，MLH1和BRCA1基因，可以很容易地实施在研究和临床实验室作为低成本的遗传筛选试验。
With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for theRB1,TP53,MLH1andBRCA1genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.