Conditions for single-strand conformation polymorphism (SSCP) analysis of BRCA1 gene using an automated electrophoresis unit.
The single-strand conformation polymorphism procedure has been applied in routine testing for hereditary diseases and cancer. However, temperature, running time, gel composition, fragment length, etc. can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes such as the breast cancer gene BRCA1. We analysed DNA samples with BRCA1 mutations in an automated system (GenePhor System; Amersham-Pharmacia Biotech, Uppsala, Sweden). The concentrations of DNA template and PCR primers, the effect of chilling after denaturation, and the temperature and time of the electrophoresis were investigated. All band-shifts were detected by electrophoresis at 5 degrees C for 2 h 15 min. Concentrations of DNA and samples used in the PCR did not affect the SSCP pattern, but chilling the PCR product in ice after denaturation was required. The type and position of mutation in the fragments did not influence the probability of a mobility shift, although SSCP analysis was more sensitive for fragments shorter than 350 bp. This automated SSCP method meets the requirements of fast turnaround and sensitivity and can be readily adapted to the screening of large genes such as BRCA1.
Campos
B
B
Servei de Genètica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
Díez
O
O
Cortés
J
J
Domènech
M
M
Pericay
C
C
Alonso
C
C
Baiget
M
M
D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D019398:Genes, BRCA1; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature
D019313:BRCA1 Protein; D017931:DNA Primers; D004273:DNA, Neoplasm