THE COVALENT ATTACHMENT OF MULTIPLE FLUOROPHORES TO DNA CONTAINING PHOSPHOROTHIOATE DIESTERS RESULTS IN HIGHLY SENSITIVE DETECTION OF SINGLE-STRANDED-DNA

被引:17
作者
CONWAY, NE [1 ]
MCLAUGHLIN, LW [1 ]
机构
[1] BOSTON COLL,DEPT CHEM,140 COMMONWEALTH AVE,CHESTNUT HILL,MA 02167
关键词
D O I
10.1021/bc00012a013
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing > 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.
引用
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页码:452 / 457
页数:6
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