Rapid method for determining the rate of DNA synthesis and cellular proliferation

A new method has been developed for determination of DNA synthesis during c ell proliferation. The method is based on the metabolism of [U-C-13(6)]gluc ose to deoxyribose (DR) and then incorporation of [U-C-13(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 C-13 enrichm ent in DR is determined in the trimethylsilyl derivative of levulinate usin g gas chromatography-mass spectrometry, The method is rapid and sensitive. It can precisely determine C-13 enrichment below 1 at.% excess in as little as 4 ng DNA, We have used this method to determine the rate of cell prolif eration in vitro and the level of DR in a given amount of DNA, The current approach has significant advantages over previously described methods and o vercomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro. (C) 2000 Academic Press.