Development of a rapid, small-scale DNA repair assay for use on clinical samples.
Double-strand breaks (DSBs) are the most lethal form of DNA damage. They can be repaired by one of two pathways, homologous recombination and non-homologous end joining (NHEJ). A NHEJ assay has previously been reported which measures joining using cell-free extracts and a linearised plasmid as DNA substrate. This assay was designed for 3 x 10(9) cells grown in vitro and utilised radioactively labelled substrate. We have scaled down the method to use smaller cell numbers in a variety of cell lines. Altering the cellular extraction procedure decreased background DNA contamination. The cleaner preparations allowed us to use SYBR Green I staining to identify joined products, which was as sensitive as 32P-end-labelled DNA. NHEJ was found in established tumour cell lines from different originating tissues, though actual levels and fidelity of repair differed. This method also allowed end joining to be assessed in clinical specimens (human blood, brain and bladder tumours) within 24 h of receiving samples. The application of this method will allow investigation of the role of DSB DNA repair pathways in human tumours.
Diggle
Christine P
CP
Cancer Research UK Clinical Centre, St James's University Hospital, Leeds LS9 7TF, UK.
Bentley
Johanne
J
Kiltie
Anne E
AE
D052160:Benzothiazoles; D002457:Cell Extracts / Q000302:isolation & purification; D002474:Cell-Free System; D002478:Cells, Cultured; D004260:DNA Repair; D003959:Diamines; D005456:Fluorescent Dyes; D005821:Genetic Techniques; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D009930:Organic Chemicals; D011804:Quinolines; D011995:Recombination, Genetic; D013997:Time Factors; D014407:Tumor Cells, Cultured
D052160:Benzothiazoles; D002457:Cell Extracts; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I