Berta Campos, Orland Díez, Joan Cortés, Montserrat Domènech, Carles Pericay, Carmen Alonso, Montserrat Baiget
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 °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.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 39, 06/2001
Pages: 401 - 404
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