Development of hapten labeled nucleotide triphosphate for nonisotopic gene detection

Abstract

Gene detection by hybridization with specific DNA probe are now extensively use in research and in routine diagnostic work. An important aspect of nucleic acid hybridization assays is the choice of substance used to label a nucleic acid probe and its ability to generate the signal. Nonisotopic labels have been the focus of development because of the limitation of radioactive labels in general application. A successful approach is to synthesize specific DNA probe from nucleotide analogues labeled with haptens, such as biotin, digoxigenin. The objective of this study is to devise a simple, convenient method of synthesis and purification of modified nucleotide using dinitrophenyl group as hapten. Two dCTP nucleotide analogues were synthesized. These were N(4)-(2,4 dinitrophenyl-6-aminohexyl)-dCTP (DNP-dCTP I) and N(4) - [ 8-(2,4 dinitrophenylamino)-l-aminooctyl-l-quinonyl-6-aminohexyl ]dCTP (DNP-dCIP II).DNP-dCTP I was synthesized by replacing the amino group of dCTP with diaminohexane by transamination reaction, and dinitrophenylation of the amino end with FDNB to give the product. DNP-dCTP II was synthesized by linking of aminohexyl dCTP and DNP-diaminooctane with p-benzoquinone. The two dinitrophenyl modified dCTP analogues of different spacer lengths were incorporated into DNA probes by standard random-primed synthesis. The incorporation of DNP-dCTP I was 53 % as effective as dCTP control. The labeled DNA probe by these two analogues were capable of detecting the same level of DNA (lO pg). The better signal could be obtained, if the optimal condition for DNA detection were further determined in more detail.