Non-isotopic detection of single-stranded conformation polymorphisms using ethidium bromide/UV light.
In the course of searching for single base substitutions resulting in polymorphisms or point mutations within the adenomatous Polyposis coli (APC) gene we established an alternative non-radioactive procedure to detect single-stranded conformation polymorphisms (SSCP) of polymerase chain reaction (PCR) products. A fast and inexpensive SSCP assay system is described in detail with general application allowing the non-isotopic identification of single base alterations of PCR products. This was achieved by combining heat denaturation of PCR products resulting in a high yield of single-stranded DNA and ethidium bromide staining of separated DNA strands in non-denaturing polyacrylamide vertical standard gels. The validity of the non-radioactive procedure described here is demonstrated by the identification of allele-specific SSCP patterns derived from intragenic polymorphisms of the APC gene.
Institut für Humangenetik, Universität, Erlangen, Germany.
W G
WG
Ballhausen
C
C
Kraus
D000483:Alleles; D001483:Base Sequence; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004996:Ethidium; D005069:Evaluation Studies as Topic; D017491:Genes, APC; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004996:Ethidium