Conformation sensitive gel electrophoresis for simple and accurate detection of mutations:: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Conformation sensitive gel electrophoresis for simple and accurate detection of mutations:: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
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DOI：
10.1073/pnas.95.4.1681
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发表时间：
1998-02-17
影响因子：
11.1
通讯作者：
Ala-Kokko, L
中科院分区：
文献类型：
--
作者：
Körkkö, J;Annunen, S;Ala-Kokko, L
以前，开发了一种称为构象敏感凝胶电泳（CSGE）的检测方法，用于对PCR产物进行采样，以确定DNA中是否存在单碱基和较大的碱基错配。该测定是基于这样的假设，即在适当的缓冲液中的轻度变性溶剂可以加重由双链DNA中的单碱基错配产生的构象变化，从而增加异源双链体和同源双链体的电泳凝胶中的差异迁移。在这里，通过将PCR产物的最大大小限制为450 bp并对PAGE条件进行几次改变，提高了CSGE测定的灵敏度。在改进的条件下，CSGE检测到了所有76个先前确定的单碱基变化，这些变化来自胶原蛋白基因的大量PCR产物，这些胶原蛋白基因包含具有高度重复和富含GC序列的多个外显子。在对736个胶原基因等位基因的调查中，CSGE检测到223个独特的单碱基错配，并通过核苷酸测序证实。CSGE与其他PCR产物扫描方法相比，具有简单、不需要特殊制备PCR产物、容量大、不使用放射性等优点。
Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for sampling PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.