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. 1992 Feb 25;20(4):871–878. doi: 10.1093/nar/20.4.871

Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP.

G Sarkar 1, H S Yoon 1, S S Sommer 1
PMCID: PMC312031  PMID: 1371869

Abstract

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 ethidium 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.

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Selected References

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