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.
Kinoshita-Kikuta
Emiko
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan.
Kinoshita
Eiji
E
Harada
Naoko
N
Koike
Tohru
T
D000241:Adenosine / Q000378:metabolism; D019313:BRCA1 Protein / Q000235:genetics*; D000080027:Cyclams; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006571:Heterocyclic Compounds / Q000737:chemistry*; D009154:Mutation; D013941:Thymine / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
D019313:BRCA1 Protein; D000080027:Cyclams; D006571:Heterocyclic Compounds; C038072:cyclen; D015032:Zinc; D000241:Adenosine; D013941:Thymine