Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation.
The accurate analysis of genetic variation has major implications in many areas of biomedical research, including the identification of infectious agents (such as parasites), the diagnosis of infections, and the detection of unknown or known disease-causing mutations. Mutation scanning methods, including PCR-coupled single-strand conformation polymorphism (SSCP), have significant advantages over many other nucleic acid techniques for the accurate analysis of allelic and mutational sequence variation. The present protocol describes the SSCP method of analysis, including all steps from the small-scale isolation of genomic DNA and PCR amplification of target sequences, through to the gel-based separation of amplicons and scanning for mutations by SSCP (either by the analysis of radiolabeled amplicons in mutation detection enhancement (MDE) gels or by non-isotopic SSCP using precast GMA gels). The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. The SSCP protocol can readily detect point mutations for amplicon sizes of up to 450-500 bp, and usually takes 1-2 days to carry out. This user-friendly, low-cost, potentially high-throughput platform has demonstrated the utility to study a wide range of pathogens and diseases, and has the potential to be applied to any gene of any organism.
Gasser
Robin B
RB
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia. robinbg@unimelb.edu.au
Hu
Min
M
Chilton
Neil B
NB
Campbell
Bronwyn E
BE
Jex
Aaron J
AJ
Otranto
Domenico
D
Cafarchia
Claudia
C
Beveridge
Ian
I
Zhu
Xingquan
X
D004252:DNA Mutational Analysis / Q000379:methods*; D023281:Genomics / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational