Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP.
Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP.
复制标题
通过 RNA 单链构象多态性 (rSSCP) 筛选突变：与 DNA-SSCP 的比较。
DOI：
10.1093/nar/20.4.871
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发表时间：
1992
影响因子：
14.9
通讯作者：
Sommer,SS
中科院分区：
文献类型：
--
作者：
Sarkar,G;Yoon,HS;Sommer,SS
单链构象多态(SSCP)是一种简单的检测DNA片段突变的方法，但检测到的突变所占比例尚不清楚。我们评价了SSCP在检测因子IX基因单碱基突变中的作用。考察了多种条件，包括电泳温度、电泳缓冲液浓度、丙烯酰胺与双丙烯酰胺的比例以及水冷和扇冷凝胶装置。根据情况，在183bp的片段中检测到12个已知突变中的10-11个，而在307bp的片段中仅检测到22个已知突变中的11-14个。我们假设单链RNA应该有更大的二级结构谱系，因为较短的发夹形成稳定的双链，并且2‘羟基可用于糖-碱和糖-糖氢键。通过将噬菌体启动子序列插入到聚合酶链式反应中，RNA-SSCP(RSSCP)可以直接与标准DNA SSCP进行比较。RSSCP总体上优于SSCP，尤其是在307bp的片段上。此外，由于rSSCP产生了丰富的转录本，因此可以通过用溴化乙酯对凝胶进行染色来快速、非放射性地检测突变。对28例血友病B患者进行了SSCP、rSSCP和基因组直接测序的盲法研究，共检测了血友病B的9个区段2.6kb的凝血因子IX基因组序列，序列范围为180~497个核苷酸。基因组直接测序检测到20个不同位点的序列变化；rSSCP检测到其中70%的变化，而SSCP只检测到35%。
Single-strand conformation polymorphism (SSCP) Is a simple method for detecting the presence of mutations in a segment of DNA, but the fraction of all mutations detected is unclear. We have evaluated SSCP for the detection of single-base mutations In the factor IX gene. Multiple conditions were examined Including electrophoresis temperature, electrophoresis buffer concentration, acrylamide to bis-acrylamide ratio, and water-cooled versus fan-cooled gel apparatuses. Depending on conditions, 10–11 of 12 known mutations were detected in a 183 bp segment whereas only 11 –14 of 22 known mutations were detected In a 307 bp segment. We hypothesized that single stranded RNA should have a larger repertoire of secondary structure because shorter hairpins form stable duplexes and the 2′ hydroxyl group is available for sugar-base and sugar-sugar hydrogen bonds. By incorporating phage promoter sequences into PCR primers, RNA-SSCP (rSSCP) could be compared directly with standard DNA SSCP. rSSCP was generally superior to SSCP, especially for the 307 bp segment. In addition, the abundance of transcript produced as a result of rSSCP allows the rapid, nonradioactive detection of mutations by staining the gel with ethldium bromide. To gauge the utility of the method In a prospective manner, a blinded study was performed in which SSCP, rSSCP, and direct genomic sequencing were compared in 28 patients with hemophilia B. A total of 2.6 kb of factor IX genomic sequence was examined in nine regions ranging from 180 to 497 nucleotides of factor IX sequence. Sequence changes at 20 different sites were detected by direct genomic sequencing; 70% of these were detected by rSSCP while only 35% were detected by SSCP.