| 摘要 |
The present invention relates to the of determining the sequence preference of DNA-RNA hybrid binding protein or its domain which comprises the following steps:
a) contacting of the purified protein or its domain, with a mixture of a library of DNA-RNA hybrids substrates, wherein DNA-RNA hybrid substrates comprise randomized sequences in the central part, preferably randomized in 9 or 10 nucleotide positions, with flanking sequences fixed and allowing a tested protein or its domain to bind the sequence to which it has an affinity;
b) separating unbound DNA-RNA hybrids by immobilization of protein or its domain, with bound DNA-RNA hybrid, to the resin, preferably glutathione agarose;
c) removing the unbound hybrids;
d) isolating the recombinant protein, together with the associated DNA-RNA hybrids,
e) amplification of the isolated hybrid using PCR, preferably RT-PCR, wherein in the amplification reaction the primers complementary to invariant sequences flanking the randomized region are used, and wherein during the PCR reaction on one of the primer the sequence of RNA polymerase promoter is added in order to obtain double-stranded DNA for in vitro transcription with RNA polymerase;
f) reverse transcription using reverse transcriptase that does not possess the RNase H activity and a DNA primer complementary to the 3' end of the RNA template in order to obtain a DNA-RNA hybrid.. |
