Multitemperature single-strand conformation polymorphism

Abstract

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 degrees C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degrees C or decreasing it by 10 degrees C from 23 degrees 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.

Rapid Differentiation of Mixed Influenza A/H1N1 Virus Infections with Seasonal and Pandemic Variants by Multitemperature Single-Stranded Conformational Polymorphism Analysis

“…Thus, the aim of our study was to develop and evaluate a new molecular method for rapid identification and discrimination of genetic variants of influenza A/H1N1 virus, including the detection of mixed infections in a single patient. The minor genetic variant detection method developed and applied in this work is based on the known multitemperature single-stranded conformational polymorphism (MSSCP) analytical and preparative capabilities (7,11). We evaluated the MSSCP approach on 2009 pandemic Table 1.…”

“…The RT-PCR products were analyzed by the MSSCP method at a strictly controlled (to Ϯ0.2°C) gel temperature in dedicated equipment, a DNAPointer system (BioVectis, Warsaw, Poland), as described by Kaczanowski et al (7). The RT-PCR products were heat denatured and resolved as single-stranded DNA (ssDNA) conformers on a 9% polyacrylamide gel under native conditions (TBE [Tris-borate-EDTA] buffer) at three different temperatures during one run.…”

P53 mutations in urinary bladder cancer patients from Central Poland

“…In this study, results of DNA sequencing of these abnormal bands from 180 E. Borkowska et al urinary cells were compared with that for genomic DNA from blood lymphocytes. The application of several combinations of polyacrylamide gels and a new option related to DNA analysis in the temperature gradients, increased the probability of detecting changes in DNA by 20-30%, as compared with the traditional SSCP (Kaczanowski et al 2001). Berggren et al (2000) applied only 2 temperature variants (in separate experiments) and 4 combinations of polyacrylamide gels to detect more than 90% of mutations in the DNA isolated from cell lines, and 100% of mutations (10/10) in samples from patients.…”

High-Throughput Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative Approach to Monitoring Drug Resistance of Hepatitis B Virus

“…The electrophoretic separation is performed under sequentially changed gel temperatures. Data in the literature show that results obtained by MSSCP are much more sensitive (0.1% of minor strains) than those obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures (23).…”