Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon |
| |
Authors: | E V Dmitrienko I A Pyshnaya D V Pyshnyi |
| |
Institution: | 1.Institute of Chemical Biology and Fundamental Medicine, Siberian Branch,Russian Academy of Sciences,Novosibirsk,Russia;2.Novosibirsk State University,Novosibirsk,Russia |
| |
Abstract: | The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic
labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short
terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed
as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and
reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for
DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm2 at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences.
It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface,
while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives
in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the
functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide
the immobilization of oligonucleotides on nylon membranes. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|