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• W2085397312 abstract "DNA damage and repair is important in both mutagenesis and cancer therapy. Study of repair mechanisms is therefore important, and quick, reliable and sensitive assays are required. At the level of individual genes, Southern blotting-based methods have proved extremely valuable (1). Recently, PCR based techniques have been developed which are equally sensitive (2,3), more rapid and use less DNA compared with Southern blotting, and allow mapping of damage and repair at the sub-gene level (∼500 bp) (4). PCR methods cannot, however, be used to measure strand-specific damage and repair. Due to the importance of transcription coupled repair this is a serious drawback. We describe a new method, strand-specific-QPCR (ss-QPCR), which allows the strand-specific measurement of DNA damage and repair in mammalian cells. The method is outlined in Figure 1 (as set up to measure lesions on the transcribed strand). DNA extracted from drug treated and untreated cells is subjected to a first round ‘linear’ PCR using a single biotinylated primer (1-tB), complementary to the transcribed strand. This PCR generates a family of single-stranded molecules some of which will be truncated due to the presence of a blocking lesion on the transcribed strand of the template DNA. All are captured on streptavidin coated paramagnetic beads and washed with NaOH to remove genomic DNA including any hybridised to the PCR products. After neutralisation, the single-stranded molecules, while still attached to the beads, serve as templates in a second, exponential, PCR. In the exponential amplification the downstream primer (primer 2) is complementary to the transcribed strand and is nested with respect to primer 1. The upstream primer (primer 3) is complementary to the non-transcribed strand and its binding site determines the length of the gene region in which damage is to be measured. In this PCR only those DNA molecules which were extended past the site of primer 3, i.e. those which were not blocked by lesions on the genomic DNA, will be exponentially amplified. Thus, provided the PCR remains in the exponential phase when stopped, the amount of product (quantified by TCA precipitation and scintillation counting) will be directly proportional to the amount of undamaged template present in the region under study of the original genomic DNA. Experiments were performed using ss-QPCR to measure damage and repair in a 350 bp region, comprising intron 1 of the human N-ras gene, after treatment of cells with the anti-cancer drug cisplatin. The primers used (Genosys, UK) were:" @default.
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• W2085397312 title "Strand-Specific Measurement of Cisplatin-Induced DNA Damage and Repair Using Quantitative PCR" @default.
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• W2085397312 doi "https://doi.org/10.1093/nar/24.5.987" @default.
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