PCR-based methods for detecting DNA damage and its repair at the sub-gene and single nucleotide levels in cells.
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.
Grimaldi
Keith A
KA
McGurk
Claire J
CJ
McHugh
Peter J
PJ
Hartley
John A
JA
D001483:Base Sequence; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000302:isolation & purification* / Q000502:physiology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000302:isolation & purification* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism; D013997:Time Factors
D004277:DNA, Single-Stranded; D009711:Nucleotides; D019914:Taq Polymerase