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A large number of chemical methods have been developed for the preparation of
nonradio labeled oligonucleotides. The chemical methods involved introduction of amino or
thiol functional group(s) at 5'-end or 3'-end of the synthetic oligonucleotides. These
functionalized oligonucleotides were then derivatized with suitable fluorescent dye in order to
prepare hybridization probes or primers for DNA sequencing, which can be detected
by nonradioisotopic methods (Agarwal, 1994, and Herdewijn, 2005). Modification at
5'-end is carried out after the synthesis of oligonucleotides. However, modification at
3'-end requires derivatization of modified solid support and subsequent oligonucleotide
synthesis. The methods so far reported for the incorporation of
3' amino functional group(s) are cumbersome (Nelson et al., 1989; Kumar et al., 1996; McMinn and Greenberg, 1998;
Stetsenko and Gait, 2001; and Mahajan et al., 2006) and require incorporation of protected amino
group in polymer support. In some previous studies, the simple procedures for the introduction
of free thiol group at 3'-end of oligonucleotides are described (Kumar, 1993a; and Kumar,
1993b). Herein, we describe an economical solid phase method for the synthesis of
3'-amino group containing oligonucleotide. This method involves the synthesis of
3-bromo-4,4'-dimethoxytrityl)-2-propanol from commercially available 3-bromo-1,2-propanediol.
Next comes the derivatization of 3-bromo-4,4'-dimethoxytrityl)-2-propanol on CPG support
via a succinyl linkage and conversion of bromo group to amino group on treatment with
ammonia during the deprotection step. The basis of this method is that the conversion of
3-bormo-1, 2-propandiol to 3'-amino-1,2-propandiol takes only 12-15 minutes, whereas deprotection
of (benzoyl and isobutyl) protected groups from oligonucleotides involves at least 2-3 h at 55
°C in 29% ammonia solution. The derivatized support is found to be fully compatible with
the established phosphoramidite chemistry of oligonucleotide synthesis. The
modified oligonucleotide synthesized using this support and their
fluorescent—conjugates—were characterized by reversed phase FPLC and UV
spectroscopy.
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