Multitemperature single-strand conformation polymorphism

Changes of gel temperature during single-strand conformation polymorphism ( SSCP) electrophoresis increase the sensitivity of mutation detection in pol ymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for singl e nucleotide polymorphism (SNP) and point mutation detection - multitempera ture single-strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1 degreesC accuracy during electropho resis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degreesC or decreasing it by 10 degrees from 23 degreesC led to the disappearance of all electrophoretic differences between five al leles of exon 8 of the human p53 gene during the SSCP analysis. The interes ting 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 o f the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost-effective tool for the s creening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.