Optimization of PCR and electrophoresis conditions enhances mutation analysis of the BRCA1 gene.
Single-strand confirmation polymorphism (SSCP) analysis on MDE gels has been extensively used to detect BRCA1 gene mutations. In screening a large cohort of ovarian cancer patients, our group was not completely satisfied with the standard published techniques. Although conventional primer sets usually amplified well, we were able to enhance band spread and resolution by varying the PCR and electrophoresis conditions for many of the individual exons. These alterations enhanced our ability to detect polymorphisms and mutations. When utilizing SSCP screening of a gene as large as BRCA1, no one set of conditions or even method may be optimal for all of the exons. Rather, a variety of different methods and conditions should be studied for each set of amplimers under analysis.
Lallas
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Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, USA.
Buller
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D002021:Buffers; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004586:Electrophoresis / Q000379:methods*; D005091:Exons; D005260:Female; D019398:Genes, BRCA1; D006801:Humans; D009154:Mutation / Q000235:genetics; D010051:Ovarian Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature; D013997:Time Factors
D002021:Buffers; D017931:DNA Primers