Affinity purification of nucleic acids and protein - nucleic acid complexes using biotinylated nucleotide probes.

摘要

This dissertation describes the development of three purification methods based on the interaction between avidin and biotin, or streptavidin and biotin, and their application to the purification of specific nucleic acid sequences and proteins associated with specific nucleic acid sequences.;The second method describes the development of a novel method for rapid plasmid library screening. The method combines the ability of recA protein to form stable complexes between linear single-stranded DNA, labeled with biotinylated nucleotides, and circular double-stranded DNA molecules, with procedures for the isolation of biotinylated nucleic acids. Using a reconstructed library, we demonstrated the selection for a specific subset of plasmids, the homologous plasmids were recovered with an average yield of 14%, with an overall enrichment of 10;The third method was developed for the isolation of protein-DNA complexes, specifically DNA replication complexes of minute virus of mice (MVM). It was shown that extracts from mouse fibroblasts infected with MVM incorporated biotinylated nucleotides into in vitro synthesized DNA. Furthermore, the replication intermediates produced in vitro were shown to be similar to intermediates seen in vivo. The incorporation of biotinylated nucleotides into newly synthesized viral DNA provided a tag which made the affinity purification of viral DNA replication complexes possible. Three different viral proteins, VP-1, VP-2, and NS-1, were shown to be specifically associated with viral DNA synthesized in vitro.;The first method presents a new procedure for the subtractive hybridization, or negative selection, of specific nucleic acid sequences. The method combines solution hybridization, using nucleic acids labeled with biotinylated nucleotides, and metal chelate chromatography. Metal chelate chromatography was shown to be an effective procedure for the reversible retention of biotinylated nucleic acids, and can be used to separate biotinylated nucleic acids and hybrids, fron non-biotinylated nucleic acids. This method was successfully used to isolate the set of unique sequences from Neisseria gonorrhoeae by subtractive hybridization with biotinylated DNA from Neisseria meningitidis, a closely related bacteria strain. The method was also used to isolate a set of cDNAs from mRNAs that were overexpressed in dimethylsulfoxide treated Friend leukemia cells.