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Multitemperature single‐strand conformation polymorphism
Author(s) -
Kaczanowski Radoslaw,
Trzeciak Lech,
Kucharczyk Krzysztof
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200109)22:16<3539::aid-elps3539>3.0.co;2-t
Subject(s) - single strand conformation polymorphism , single nucleotide polymorphism , microbiology and biotechnology , polymorphism (computer science) , gel electrophoresis , exon , polymerase chain reaction , snp , biology , point mutation , polyacrylamide gel electrophoresis , dna , genetics , allele , gene , genotype , mutation , biochemistry , enzyme
Changes of gel temperature during single‐strand conformation polymorphism (SSCP) electrophoresis increase the sensitivity of mutation detection in polymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for single nucleotide polymorphism (SNP) and point mutation detection – multitemperature single‐strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1°C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8°C or decreasing it by 10°C from 23°C led to the disappearance of all electrophoretic differences between five alleles of exon 8 of the human p53 gene during the SSCP analysis. The interesting result was the detection of two additional SNPs (out of seven analyzed) in exon 7 of the human PAH gene during a one hour MSSCP electrophoresis. This result is better than that obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost‐effective tool for the screening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.