Mutational analysis using denaturing gradient gel electrophoresis and PCR.
Denaturing gradient gel electrophoresis (DGGE) separates (DNA) molecules based on their sequence. Using the proper conditions, all base-pair substitutions can be resolved from the wild-type sequence using DGGE. Polymerase chain reaction (PCR) permits rapid amplification of a given region of the genome. In this paper, we demonstrate the utility of DGGE combined with PCR for mutation analysis by presenting different examples: (i) analysis of mouse p53 cDNA for mutations, (ii) simultaneous analysis of thousands of 4NQO-induced mutants for mutations in HPRT exon 3, (iii) examination of the fidelity of the thermostable DNA polymerase isolated from Pyrococcus furiosus (Pfu), (iv) purification of mutant DNA from contaminating wild-type DNA from mouse spleenic T-cell clones.
Cariello
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University of North Carolina, Pathology Department, Chapel Hill 27599.
Skopek
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TR
D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity; D000818:Animals; D001105:Archaea / Q000201:enzymology; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000187:drug effects; D004252:DNA Mutational Analysis / Q000379:methods*; D004277:DNA, Single-Stranded / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D016158:Genes, p53; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes; D019914:Taq Polymerase; D013866:Thioguanine / Q000494:pharmacology
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D015112:4-Nitroquinoline-1-oxide; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D013866:Thioguanine