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A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

✍ Scribed by William J. Bodell; Jytte Rasmussen

Publisher: Elsevier Science
Year: 1984
Tongue: English
Weight: 299 KB
Volume: 142
Category: Article
ISSN: 0003-2697
DOI: 10.1016/0003-2697(84)90501-3

No coin nor oath required. For personal study only.

✦ Synopsis

Randerath's procedure for 32P postlabeling of 3'monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'Monophosphate deoxyribonucleotides are converted to 3',S-bis["*P]phosphate deoxyribonuclcotides with polynucleotide kinase and ['*P]ATP; these products are enzymatically converted by PI nuclease and polynucleotide kinase into S-['*P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0. I M NaH2POI, pH 6.5. Labeled mononucleotides in the elIluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.

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