PCR-based methods for detecting DNA damage and its repair at the sub-gene and single nucleotide levels in cells

• Published in: Molecular biotechnology Vol. 20; no. 2; pp. 181 - 196
• Main Authors: GRIMALDI, Keith A, MCGURK, Claire J, MCHUGH, Peter J, HARTLEY, John A
• Format: Journal Article
• Language: English
• Published: Totowa, NJ Humana Press 01.02.2002; Springer Nature B.V
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cells; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - physiology; DNA Repair - physiology; DNA, Single-Stranded - isolation & purification; DNA, Single-Stranded - physiology; Fundamental and applied biological sciences. Psychology; Genes; Models, Chemical; Molecular Sequence Data; Nucleotides - isolation & purification; Nucleotides - physiology; Polymerase chain reaction; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Studies; Taq Polymerase - metabolism; Time Factors
• ISSN: 1073-6085; 1559-0305; 1073-6085
• DOI: 10.1385/MB:20:2:181

Three PCR-based methods are described that allow covalent drug-DNA adducts, and their repair, to be studied at various levels of resolution from gene regions to the individual nucleotide level in single copy genes. A quantitative PCR (QPCR) method measures the total damage on both DNA strands in a gene region, usually between 300 and 3,000 base pairs in length. Strand-specific QPCR incorporates adaptations that allow damage to be measured in the same region as QPCR but in a strand-specific manner. Single-strand ligation PCR allows the detection of adduct formation at the level of single nucleotides, on individual strands, in a single copy gene in mammalian cells. If antibodies to the DNA adducts of interest are available, these can be used to capture and isolate adducted DNA for use in single-strand ligation PCR increasing the sensitivity of the assay.