Mutation analysis of genetic diseases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis.
The Single Strand Conformation Polymorphism (SSCP) technique is widely used in mutation analysis. We have introduced several modifications to the SSCP method, which overcome the problem of incomplete denaturation or reannealing of DNA during electrophoresis. The modifications consist of asymmetrical PCR amplification of the sequence of interest, electrophoresis with a higher concentration of acrylamide, and the analysis of the DNA fragments under u.v. light. We have applied this method to the analysis of two specific diseases: neurofibromatosis type 1 (NF1) and cystic fibrosis (CF) from PCR amplified exons. Two single nucleotide changes were observed with this method.
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004277:DNA, Single-Stranded / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004996:Ethidium / Q000528:radiation effects; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D009456:Neurofibromatosis 1 / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D013194:Staining and Labeling; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D004996:Ethidium