Zinc(II)-cyclen polyacrylamide gel electrophoresis for detection of mutations in short Ade/Thy-rich DNA fragments.
We describe an improved gel-based method with an additive Zn(2+)-cyclen complex (cyclen, 1,4,7,10-tetraazacyclododecane), Zn(2+)-cyclen-PAGE, for mutation detection in DNA fragments by PCR that contain more than 65% Ade/Thy bases and fewer than 100base pairs (bp). Existing techniques have a problem in analyzing such short Ade/Thy-rich fragments because the duplexes are disrupted and are not detectable due to binding of Zn(2+)-cyclen to Thy bases. In this strategy using a PCR primer with a Gua/Cyt-lined sequence attached at its 5'-end, we successfully detected a mutation in an 86-bp Ade/Thy-rich region of the BRCA1 gene from formalin-fixed paraffin-embedded breast cancer-tissue sections.