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QUANTIFICATION OF PYRIMIDINE DIMERS AND APURINIC SITES IN DNAs OF UNIFORM LENGTH
Author(s) -
Ciomei Marina,
Sutherland Betsy M.,
Ciarrocchi Giovanni
Publication year - 1984
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1984.tb03895.x
Subject(s) - ap site , dna , agarose , endonuclease , agarose gel electrophoresis , dna supercoil , molecule , pyrimidine dimer , chemistry , pyrimidine , gel electrophoresis , biophysics , depurination , electrophoresis , gel electrophoresis of nucleic acids , dna damage , stereochemistry , biochemistry , biology , dna replication , organic chemistry
— A new simple in vitro assay for the determination of pyrimidine dimers and/or apurinic/apyrimidinic sites in non‐radioactive DNA has been developed. In this procedure, DNA substrates of uniform length‐which may be supercoiled, partially relaxed, relaxed or linear‐are treated with agents which produce specific single strand nicks at the site of the lesion. The number of lesions per molecule can be expressed as a function of the amount of single‐stranded molecules left intact after the specific nicking treatment. Unreacted molecules, which retain the original uniform length, are separated from the other smaller reaction products by electrophoresis on an alkaline agarose gel. In the case of circular molecules, the substrate is linearized by the use of an appropriate restriction endonuclease before loading on the gel. The amount of intact DNA molecules is obtained by integrating the corresponding peak of absorption after densitometric scan of the negative of the gel picture. This assay can detect up to eight damaged sites per duplex molecule. This method could be particularly useful when dealing with mixtures of DNA with different degrees of supercoiling or for comparisons between linear and circular DNA substrates.