Comparison of enzyme mismatch cleavage and chemical cleavage of mismatch on a defined set of heteroduplexes.
Two mutation detection methods based on the cleavage of mismatched heteroduplexes were compared and evaluated. These techniques, chemical cleavage of mismatch (CCM) and enzyme mismatch cleavage (EMC), have the advantages over other available methods of being able to detect and localize mutations in relatively large fragments of DNA (> or = 1 kb). We have constructed clones that enable us to create heteroduplexes of 500 bp, 1 kb, and 1.5 kb and have assessed each of the methods over a range of criteria. Both were able to detect and localize all four types of single-base mismatch and insertion/deletions of 1-5 bp. Whereas EMC was efficient at detection of insertion/deletions in a broad size range of fragments and has the advantage over CCM of using no hazardous chemicals, in our hands it has not been sufficiently robust that we felt confident to consider it for diagnostic use in its present form. CCM using hydroxylamine was efficient over the entire range of fragment sizes tested and using potassium permanganate with tetraethylammonium chloride was efficient up to 1 kb.
Deeble
V J
VJ
Yorkshire Regional DNA Laboratory, Leeds, UK.
Roberts
E
E
Robinson
M D
MD
Woods
C G
CG
Bishop
D T
DT
Taylor
G R
GR
D020029:Base Pairing; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D004252:DNA Mutational Analysis; D004706:Endodeoxyribonucleases / Q000494:pharmacology*; D005069:Evaluation Studies as Topic; D019398:Genes, BRCA1; D005820:Genetic Testing / Q000379:methods*; D020180:Heteroduplex Analysis; D006801:Humans; D019811:Hydroxylamine / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology*; D017354:Point Mutation; D011196:Potassium Permanganate / Q000494:pharmacology; D017384:Sequence Deletion; D019789:Tetraethylammonium / Q000494:pharmacology
D009153:Mutagens; D011196:Potassium Permanganate; D019811:Hydroxylamine; D019789:Tetraethylammonium; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII