Differential incorporation of biotinylated nucleotides by terminal deoxynucleotidyl transferase.

Synthetic oligonucleotides prepared from known DNA or amino acid sequences can be used as gene probes for detection of DNA and RNA. Short oligonucleotide probes can discriminate between two sequences which differ by only a single nucleotide (1). Biotin is the most frequently used non-radioactive reporter group for labelling of nucleic acids. Enzymatic and/or chemical synthesis have been used to label oligonucleotides with biotin (for review see reference 2). More recently, biotinylated probes have been prepared with one or more biotinylated nucleotides at the 3' end using DNA polymerase (3) or Terminal deoxynucleotidyl Transferase (TdT) (4). In this paper, we have compared the efficiency of labelling synthetic oligonucleotides with biotinylated nucleotides using TdT. We have compared the use of biotinylated dCTP and dATP analogs for incorporation by TdT. Furthermore, we have shown the utility of these probes in chemiluminescent detection of nucleic acids immobilized on solid support. To study the efficiency of labelling oligonucleotides with TdT, a 22-base oligonucleotide specific for the Herpes Simplex virus thymidine kinase gene was synthesized (5' CCC GAG CCG ATG ACT TAC TGC C 3'). The oligonucleotide was labelled with P at the 5' end using T4 polynucleotide kinase (Life Technologies, Inc.). The P-labelled oligonucleotide was mixed with 1 nmole of unlabelled oligonucleotide and was used as a substrate for tailing with biotinylated nucleotides (Gibco BRL). The reaction products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. We compared the efficiency of incorporation of N-(Nbiotinyl-6-aminohexyl)dATP (biotin-7-dATP),N-[N-(Nbiotinyl-e-aminocaproyl)-6-aminohexyl]dATP (biotin14-dATP), N(N-biotinyl-6-aminohexyl)dCTP (biotin-7-dCTP), N-[N-(Nbiotinyl-e-aminocaproyl)-6-aminohexyl]dCTP (biotin14-dCTP), and 5-[N-(N-biotinyl-e-aminocaproyl)-3-aminoallyl]dUTP (biotin-11-dUTP). Biotinylated derivatives of dCTP and dUTP consistently showed better incorporation with TdT than their dATP counterparts. Using biotinylated dCTP derivatives the majority of the substrate molecules were tailed with multiple biotinylated residues (fig. 1). With biotin-11-dUTP as the nucleotide, the majority of the molecules were tailed; however the number of biotinylated residues added was limited to one (data not shown).