An oligodeoxynucleotide, 5'CTCTCTCTCTCTCTCT3' was synthesized on disulphide bond containing nylon beads and membranes supports. The protecting groups form the oligodeoxynucleotide was deblocked by ammonia treatment. The 5'CTCTCTCTCTCTCT3' remained anchored on to the nylon support was used for duplex and triplex hybridization studies with biotinylated human DNA. The hybridization experiments showed that biotinylated human DNA hybridized with support bound oligonucleotide as the color of the nylon support became purple on development with BCIP + NBT substrate. The hybridization experiment with underivatized nylon membrane strip (2 mm ´ 5 mm) showed no color. However, blank experiment was carried out using controlled pore glass support (CPG-500 A^o) which showed purple color. Hence, nylon membrane strips bound oligonucleotides can be used as a diagnostic probe and is suitable for colorimetric assays unlike CPG support.

Dot hybridization (Maniatis et al., 1982), colony hybridization (Gruntein and Hogness, 1975) and hybridization technique (Sourthern, 1975) have widely been used in molecular biology. These methods are based on noncovalent immobilization of nucleic acids on matrices, such as nitrocellulose filter and nylon membranes. Use of solid supports in DNA probe-based hybridization was reviewed (Meinkoth and Wahl, 1984). The oligonucleotides were attached to supports for hybridization with target DNA (Ghosh and Mosso, 1987). The synthesis of oligonucleotides on glass plates and ballotini beads, used for hybridization, obviating the need for detaching them from the matrices on which they were synthesized and their reattachment to the hybridization support was reported (Maskos and Sourthen, 1992). However, oligonucleotides were synthesized manually on glass matrices. We have earlier reported the synthesis, linker stability and hybridization properties of disulphide and urethane linkage containing controlled pore glass (Kumar, 1994). Nylon beads are readily available and are much cheaper than the other supports used for the oligonucleotide synthesis. Nylon beads have successfully been used for nonradioisotope hybridization assay (Ness et al., 1991). Nylon beads are small beads of nylon and have not used for the synthesis of oligonucleotides earlier. Nylon membranes are frequently used in the hybridization studies because of their low nonspecific binding properties. Recently, we have described a method to derivatize thiolated nylon beads and membranes strips (3 mm ´ 3 mm ´ 2 mm, 15 mg and 2 mm ´ 5 mm) with 4, 4'-dimethoxytrityl-6-mercaptohexane for the synthesis of oligonucleotides using automated gene assembler (Pharmacia) (Kumar, 2010). Earlier, we have shown that triplex hybridization yield is 5-6 times more than the duplex hybridization yield (Kumar, 1994). In this paper, we show the duplex and triplex hybridization studies with biotinylated human DNA using oligonucleotide (5'CTCTCTCTCTCTCT3') anchored on to the nylon support. The hybridization experiments showed that biotinylated human DNA hybridized with BCIP + NBT substrate. The blank hybridization experiment with underivatized nylon support showed no color. However, blank experiment was carried out using controlled pore glass support (CPG-500 A^o) showed purple color. This clearly demonstrated that nylon supports bound oligonucleotides can be used as a diagnostic probe.

Biotechnology Journal, Oligonucleotides, Hybridization Experiments, Dot Hybridization, Colony Hybridization, Molecular Biology, Nonradioisotope Hybridization, Duplex Hybridization Experiment, Triplex Hybridization Experiment, Nylon Membrane Strips, Biotinylated Human Genomic DNA.