Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Numerous deoxyribonucleoside triphosphate (dNTP) assays utilizing DNA polymerase exist.
• Existing methods require critical evaluation of substrates, enzyme purity, reaction conditions, and sample interference.
• Accurate dNTP quantification is crucial for understanding DNA replication and repair.

Purpose of the Study:

• To critically evaluate and optimize a deoxyribonucleoside triphosphate assay using DNA polymerase.
• To improve the sensitivity, range, accuracy, and reproducibility of dNTP measurements.
• To determine the dNTP content in Chinese hamster ovary K-1 cells using the refined assay.

Main Methods:

• Critically evaluated all aspects of the DNA polymerase-based dNTP assay.
• Utilized a phosphatase-free DNA polymerase preparation and 2'-dAMP to inhibit exonuclease activity.
• Implemented corrections for background and sample incorporation, accounting for radioactive precursor dilution.

Main Results:

• Developed a refined dNTP assay with improved accuracy and reproducibility.
• Reported sensitivity, range, and accuracy values for the optimized method.
• Quantified the deoxyribonucleoside triphosphate content in Chinese hamster ovary K-1 cells.

Conclusions:

• The optimized dNTP assay provides a reliable method for cellular dNTP quantification.
• The refined method addresses common sources of error, including enzyme purity and sample effects.
• The reported dNTP values for CHO K-1 cells serve as a benchmark for future studies.