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Measuring DNA synthesis rates with [1-13C]glycine
1Department of Pharmacology, George Washington University, School of Medicine and Health Sciences, Washington, DC 20037, USA.
Analytical Chemistry
|May 26, 1998
Summary
This study introduces a new stable-isotopic method using [1-13C]-glycine to measure DNA synthesis rates. This nonradioactive approach accurately tracks purine incorporation in cells, offering a sensitive alternative to traditional methods.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Accurate measurement of DNA synthesis rates is crucial for understanding cell proliferation and disease.
- Existing methods often involve radioactive tracers, posing safety and disposal concerns.
Purpose of the Study:
- To develop and validate a stable-isotopic method for measuring DNA synthesis rates.
- To utilize [1-13C]-glycine as a tracer for de novo purine biosynthesis.
- To establish a nonradioactive, nontoxic alternative for DNA synthesis rate determination.
Main Methods:
- Human hepatoma (HEP G2) cells were cultured with [1-13C]-glycine.
- DNA was extracted, hydrolyzed to nucleosides, and separated using High-Performance Liquid Chromatography (HPLC).
- The HPLC effluent was analyzed using a chemical reaction interface coupled with an isotope ratio mass spectrometer (HPLC/CRI/IRMS).
Main Results:
- The isotope ratio of deoxythymidine (a pyrimidine) remained unchanged, as expected.
- Deoxyadenosine and deoxyguanosine (purines) showed enrichment in 13C, reflecting cellular growth.
- The method accurately determined cell doubling times, consistent with established techniques like tritiated thymidine incorporation and direct cell counts.
Conclusions:
- The [1-13C]-glycine and HPLC/CRI/IRMS method is a sensitive and selective approach for measuring DNA synthesis rates.
- This stable-isotopic technique provides a reliable, nonradioactive, and nontoxic alternative to current methods.
- The findings support the utility of this method for various research applications in cell biology and cancer research.