Quantitation of Gene-Specific DNA Damage by Competitive PCR

✍ Scribed by Lawrence P. Fernando; Philip J. Kurian; Melihat Fidan; Daniel J. Fernandes

• Book ID: 102563986
• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 223 KB
• Volume: 306
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5705

✦ Synopsis

A sensitive assay for quantitating DNA damage within individual genes would be a valuable tool for identifying the molecular mechanisms of disease and the sites of action of various carcinogens and anticancer drugs. This report describes a competitive PCR assay that was used to quantitate DNA damage induced by anticancer drugs within a 683-bp region of the c-myc gene in human CEM leukemia cells. Absolute quantitation of gene-specific DNA damage (attomoles or molecules of damaged DNA sequences) was achieved by coamplification of a homologous internal standard that has the same primer binding sites and PCR amplification efficiency as c-myc. The variability (standard error) associated with four separate determinations of the amount of c-myc sequence in 300 ng of DNA from untreated cells (6.80 +/- 0.05 SE amol) was less than 1% of the mean. The assay was capable of quantitating direct DNA damage that was induced by therapeutic concentrations of VM-26 and cisplatin prior to the onset of cellular apoptosis or necrosis. Both VM-26 (1-10 microM) and cisplatin (25-100 microM) induced a dose-dependent decrease in the amount of intact c-myc sequence. This assay should be readily adaptable to current real-time PCR protocols.