'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses.
A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.
Hongyo
T
T
Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD.
Buzard
G S
GS
Calvert
R J
RJ
Weghorst
C M
CM
D000818:Animals; D002021:Buffers; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016158:Genes, p53; D011905:Genes, ras; D006801:Humans; D007201:Indicator Dilution Techniques; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D051381:Rats; D013696:Temperature
D002021:Buffers; D004277:DNA, Single-Stranded