Enzymatic mutation detection. Phosphate ions increase incision efficiency of endonuclease VII at a variety of damage sites in DNA.
The ability of endonuclease VII (endo VII) to cleave at mispairings in double-stranded DNA has recently been used for enzymatic mutation detection (EMD) [R. Youil, B.W. Kemper, R.G.H. Cotton, Proc. Natl. Acad. Sci. USA 92 (1995) 87-91]. The method is based on mapping cleavages in heteroduplex DNAs obtained from mutant and wildtype sequences. Despite the capability of endo VII to cleave at all possible mispairings, relative cleavage efficiencies vary considerably for individual mismatches and may escape detection if located in an unfavorable sequence surrounding. We report here improved reaction conditions which can increase the selectivity of the enzyme for mismatches up to 500-fold, as demonstrated with a mutation in a 247 nt long fragment from exon 7 of human gene p53. The new conditions involve replacement of Tris/HCl buffer by phosphate buffer and change from pH 8.0 to 6.5. Various concentrations of phosphate ions should be tried in the assay to meet individual requirements of the substrate.
Golz
S
S
Institut für Genetik, Universität zu Köln, Germany.
Greger
B
B
Kemper
B
B
D001482:Base Composition; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D016158:Genes, p53; D006801:Humans; D010710:Phosphates / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity / Q000187:drug effects
D010710:Phosphates; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII