Rapid method for determining the rate of DNA synthesis and cellular proliferation.
A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.
Nissim
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I
Division of Child Development and Rehabilitation Medicine, University of Pennsylvania School of Medicine, Abramson Pediatric Research Center, Philadelphia, Pennsylvania 19104-4318, USA.
Starr
S E
SE
Sullivan
K E
KE
Campbell
D E
DE
Douglas
S D
SD
Daikhin
Y
Y
Yudkoff
M
M
D001681:Biological Assay / Q000379:methods*; D002455:Cell Division; D004261:DNA Replication; D006801:Humans; D012680:Sensitivity and Specificity; D020298:U937 Cells