Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA.
A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.
D044503:5-Methylcytosine; D001435:Bacteriophages / Q000235:genetics; D002855:Chromatography, Thin Layer / Q000379:methods; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis; D004247:DNA / Q000032:analysis*; D045647:DNA Glycosylases; D004279:DNA, Viral / Q000032:analysis; D003856:Deoxyuracil Nucleotides / Q000032:analysis; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D010920:Placenta / Q000032:analysis; D011067:Poly dA-dT / Q000032:analysis*; D011089:Polydeoxyribonucleotides / Q000032:analysis*; D011094:Polyethyleneimine; D013447:Sulfites; D014498:Uracil / Q000032:analysis*; D051981:Uracil-DNA Glycosidase
D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D011089:Polydeoxyribonucleotides; D013447:Sulfites; D011067:Poly dA-dT; D014498:Uracil; D044503:5-Methylcytosine; D003596:Cytosine; D011094:Polyethyleneimine; D004247:DNA; C007267:2'-deoxyuridylic acid; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase