Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: a fast, simple method for detection of common mutations in BRCA1 and BRCA2.
As a result of intensive studies on hereditary breast and ovarian cancers, two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified. In each gene, a small number of specific mutations have been found at relatively high frequency in certain ethnic populations. The mutations, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2, have been identified as common mutations in the Ashkenazi Jewish population, with a combined frequency of 2.0 to 2.5%. Women who have one of the above three common mutations are at a high risk of developing breast or ovarian cancer. Consequently, accurate and cost-effective detection of these three mutations may have important implications for risk assessment in susceptible women and men. In this report, we describe a fast and simple capillary electrophoresis (CE)-based method using a polymer network for screening the three common mutations in BRCA1 and BRCA2. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify three DNA fragments of 258, 296, and 201 bp for detection of the 185delAG, 5382insC, and 6174delT mutations, respectively. After the PCR products were denatured, a single-strand conformation polymorphism (SSCP) profile could be obtained for each mutation in less than 10 min by CE in a polymer network. We demonstrate the potential provided by translating this assay to the microchip format where the SSCP analysis is complete in 120 s, representing only a fraction of the reduction in analysis time that can be achieved with microchip technology. The speed and simplicity of the SSCP methodology for detection of these mutations make it attractive for use in the clinical diagnostic laboratory.
Tian
H
H
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Jaquins-Gerstl
A
A
Munro
N
N
Trucco
M
M
Brody
L C
LC
Landers
J P
JP
D019313:BRCA1 Protein / Q000097:blood / Q000235:genetics*; D024682:BRCA2 Protein; D004252:DNA Mutational Analysis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D014157:Transcription Factors / Q000097:blood / Q000235:genetics*
D019313:BRCA1 Protein; D024682:BRCA2 Protein; D005819:Genetic Markers; D009363:Neoplasm Proteins; D014157:Transcription Factors