Direct genomic multiplex PCR for BRCA1 and application to mutation detection by single-strand conformation and
1Department of Physiology, University of Utah Health Sciences Center, Salt Lake City, Utah 84108-1270, USA. david.f.barker@m.cc.utah.edu
Human Mutation
|October 3, 2000
Summary
Multiplex PCR effectively amplifies large genes like BRCA1 for mutation screening. This method efficiently detects genetic variations and facilitates haplotype analysis for improved genetic testing.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- Mutation detection methods often rely on PCR amplification.
- Multiplex PCR enhances screening efficiency but faces challenges with large genes like BRCA1.
- BRCA1 presents difficulties due to its extensive coding regions, numerous exons, and varied mutations.
Purpose of the Study:
- To apply a general multiplex PCR approach for efficient screening of the BRCA1 gene.
- To develop a robust method for amplifying all BRCA1 coding regions and promoter segments.
- To identify and characterize novel polymorphisms within the BRCA1 gene.
Main Methods:
- Utilized fifteen triplex PCRs and one single PCR condition for comprehensive BRCA1 amplification.
- Employed Single-Strand Conformation Polymorphism (SSCP)/Heteroduplex (HDX) gel electrophoresis for mutation analysis.
- Developed a triplex PCR assay for detecting specific BRCA1 deletion polymorphisms and haplotypes.
Main Results:
- Successfully amplified all BRCA1 coding regions and promoter segments.
- Detected mobility differences in all 34 sets of allelic BRCA1 fragments analyzed.
- Identified a novel compound deletion polymorphism (CTTCT(4)CT(10)CT(12) >CT(4)CT(11)) in IVS7, a 15 bp net deletion.
- Demonstrated this deletion is a common polymorphism defining major BRCA1-RNU2 haplotypes globally.
Conclusions:
- The described multiplex PCR approach is effective for robust amplification of large genes like BRCA1.
- This method allows for rapid development of efficient mutation testing strategies.
- The approach is generally applicable for screening diverse mutations in genes with large coding regions or multiple exons.
