Novel chemical method for the preparation of nucleic acids for nonisotopic hybridization.
A novel chemical method was used to prepare biotin-labeled nucleic acids for nonisotopic hybridization. The method involves the transamination of unpaired cytosine residues in polynucleotides with sodium bisulfite and ethylenediamine. Primary amino groups on the cytosine derivatives are then reacted with biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester. Biotinylated probes hybridized with 1 to 2 pg of nitrocellulose filter-bound DNA and were visualized with a colorimetric detection technique. This method is simpler and less expensive than other methods for the preparation of nonisotopic probes. In addition, it is more versatile since the chemically modified bases can potentially react with other "indicator" molecules or proteins such as an enzyme. The specificity for unpaired cytosine residues is another advantage which could allow for the selective labeling of a specific region of a double-stranded nucleic acid. This improved labeling method should lead to the wider application of hybridization techniques in diagnostic microbiology and basic research in infectious diseases.
D000260:Adenoviruses, Human / Q000235:genetics; D000614:Aminocaproates; D010582:Bacteriophage lambda / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives; D003596:Cytosine; D004279:DNA, Viral / Q000032:analysis*; D005029:Ethylenediamines; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D011119:Polynucleotides / Q000235:genetics; D013388:Succinimides; D013447:Sulfites
D000614:Aminocaproates; D004279:DNA, Viral; D005029:Ethylenediamines; D007202:Indicators and Reagents; D011119:Polynucleotides; D013388:Succinimides; D013447:Sulfites; C031234:ethylenediamine; D001710:Biotin; C048592:biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester; D003596:Cytosine; C009279:sodium bisulfite