PCR conditions and DNA denaturants affect reproducibility of single-strand conformation polymorphism patterns for BRCA1 mutations.

Abstract

Single-strand conformation polymorphism (SSCP) is a mutation screening method based on changes in the secondary structure(s) in a defined single-stranded DNA fragment caused by a change in sequence (1). These structural changes are detected as alterations in the fragment mobility by native gel electrophoresis. The method is one of the easiest screening procedures to perform, is very cost effective, requires a minimum amount of equipment, and has a reportedly high mutation detection rate (95% for some genes ((2)) ). Although SSCP is fast and simple, problems with the reproducibility of the technique have been a major hindrance. Several variables of gel electrophoresis conditions have been tested (2)(3)(4), but studies have focused on the detection of mutations and not on the reproducibility of the SSCP band pattern. Analytical steps performed before electrophoresis may affect this reproducibility. The variables can be classified into two groups: PCR reaction conditions and post-PCR conditions. Neither of these variables have been adequately investigated. We investigated the concentrations of MgCl2, the DNA template, and the PCR primers in the PCR reaction, as well as the choice and concentrations of chemical denaturants. Eleven DNA samples from patients with characterized mutations in the breast cancer gene BRCA1 were obtained from Coriell Cell Repositories (Camden, NJ). The Coriell catalog numbers for the DNA samples were NA13708–15, NA14090, NA14093, and NA14097. Samples of DNA specimens from women with no known history of breast cancer were used as controls and were collected according to institutional guidelines approved by the Committee on Human Ethics. PCR products were obtained using Ready-To-Go PCR beads (Amersham-Pharmacia Biotech). In our standard 25-μL reaction volume, 50 ng of genomic DNA and 50 ng of each primer were used. To amplify the BRCA1 mutation Glu1250ter, we used the primer set 11.15 of …