Gel-based nonradioactive single-strand conformational polymorphism and mutation detection: limitations and solutions.
Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every PCR reaction product and assisting in choosing only the amplicons of interest with expected mutation. The principle of detection of small changes in DNA sequences is based on the changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence-dependent. The limitations faced in SSCP range from the routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems could be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP continues to remain the method of choice to analyze mutations and SNPs in order to understand genomic variations, spontaneous and induced, and the genetic basis of diseases.
Gupta
Vibhuti
V
National Centre of Applied Human Genetics, Jawaharal Nehru University, Delhi, India.
Arora
Reetakshi
R
Ranjan
Anand
A
Bairwa
Narendra K
NK
Malhotra
Dheeraj K
DK
Udhayasuriyan
P T
PT
Saha
Anjana
A
Bamezai
Ramesh
R
D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA / Q000379:methods*