Detection of 8-hydroxyguanine in small amounts of DNA by 32P postlabeling.
A method for the sensitive detection of 8-hydroxyguanine residues in small amounts of DNA (0.2-2 micrograms) was developed. It comprises (i) the enzymatic hydrolysis of DNA to 2'-deoxyribonucleotide 3'-monophosphates, (ii) degradation of the bulk amount of normal purine and pyrimidine deoxyribonucleotides in the DNA digest by treatment with trifluoroacetic acid and hydrazine, respectively, under conditions retaining the structure of d(8-OH-G)p necessary for 5' phosphorylation by T4 polynucleotide kinase (PNK), (iii) 5' phosphorylation of d(8-OH-G)p by T4 PNK-catalyzed transfer of 32P from [gamma-32P]ATP, and (iv) 2D thin-layer chromatography on polyethyleneimine-cellulose sheets to purify and resolve 32P-postlabeled d(8-OH-G)p. Model experiments with mixtures composed of synthesized d(8-OH-G)p and DNA hydrolysate indicate that it is possible to detect one 8-hydroxyguanine residue out of 2 x 10(6) normal bases starting with 1 microgram DNA. The methodology, which allows for a further decrease of this detection limit, might be very useful for the sensitive detection of DNA damage induced by activated oxygen species in small amounts of DNA. We demonstrate the formation of 8-OH-G in DNA in vitro by low doses of 60Co gamma-rays.
Lutgerink
J T
JT
Division of Chemical Carcinogenesis, The Netherlands Cancer Institute, Amsterdam.
de Graaf
E
E
Hoebee
B
B
Stavenuitez
H F
HF
Westra
J G
JG
Kriek
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; C024829:8-hydroxyguanine; D006147:Guanine; D000255:Adenosine Triphosphate; D004247:DNA