Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation.
Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.
Quaite
F E
FE
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Sutherland
J C
JC
Sutherland
B M
BM
D004247:DNA / Q000302:isolation & purification / Q000528:radiation effects*; D004249:DNA Damage; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004706:Endodeoxyribonucleases; D000455:Medicago sativa / Q000528:radiation effects*; D016982:Micrococcus luteus / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes; D009699:N-Glycosyl Hydrolases; D013472:Sunlight; D014466:Ultraviolet Rays
D009097:Multienzyme Complexes; C032219:UV endonuclease; D004247:DNA; D004706:Endodeoxyribonucleases; D009699:N-Glycosyl Hydrolases