Nucleic acid hybridization techniques have a wide application in the biological sciences for the identification and quantification of specific nucleotide sequences in complex mixtures of nucleic acids. Most current hybridization assay systems use polynucleotide probes labeled with radioisotopes. Unpaired cytosine residues on deoxyribonucleic acids can be modified by the addition of sodium bisulfite to the 5,6-double bond of the pyrimidine base. The exocyclic amino group of the cytosine–bisulfite adducts can undergo substitution with various amines in the presence of ethylenediamine, transamination forms an N-substituted cytosine derivative that has a side chain terminating in a primary amino group. The extent of formation of the N4-substituted cytosine residue varies from 12 to 56%, depending on the pH and bisulfite concentration of the reaction mixture. The efficiency of the reaction between bisulfite–ethylenediamine-modified cytosine residues and N-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester ranges from 77% for the most extensively modified nucleic acids to 100% for the lightly modified nucleic acids.
ASJC Scopus subject areas
- Molecular Biology