Abstract
We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.
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- Fargnoli J., Holbrook N. J., Fornace A. J., Jr Low-ratio hybridization subtraction. Anal Biochem. 1990 Jun;187(2):364–373. doi: 10.1016/0003-2697(90)90471-k. [DOI] [PubMed] [Google Scholar]
- Hakvoort T. B., Leegwater A. C., Michiels F. A., Chamuleau R. A., Lamers W. H. Identification of enriched sequences from a cDNA subtraction-hybridization procedure. Nucleic Acids Res. 1994 Mar 11;22(5):878–879. doi: 10.1093/nar/22.5.878. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Honigberg S. M., Rao B. J., Radding C. M. Ability of RecA protein to promote a search for rare sequences in duplex DNA. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9586–9590. doi: 10.1073/pnas.83.24.9586. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson A. F., Wang R., Ji H., Chen D., Guilfoyle R. A., Smith L. M. Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture. Anal Biochem. 1996 Feb 1;234(1):83–95. doi: 10.1006/abio.1996.0053. [DOI] [PubMed] [Google Scholar]
- Liang P., Pardee A. B. Recent advances in differential display. Curr Opin Immunol. 1995 Apr;7(2):274–280. doi: 10.1016/0952-7915(95)80015-8. [DOI] [PubMed] [Google Scholar]
- Lisitsyn N. A. Representational difference analysis: finding the differences between genomes. Trends Genet. 1995 Aug;11(8):303–307. doi: 10.1016/s0168-9525(00)89087-3. [DOI] [PubMed] [Google Scholar]
- McClelland M., Mathieu-Daude F., Welsh J. RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet. 1995 Jun;11(6):242–246. doi: 10.1016/s0168-9525(00)89058-7. [DOI] [PubMed] [Google Scholar]
- Radding C. M. Helical interactions in homologous pairing and strand exchange driven by RecA protein. J Biol Chem. 1991 Mar 25;266(9):5355–5358. [PubMed] [Google Scholar]
- Rigas B., Welcher A. A., Ward D. C., Weissman S. M. Rapid plasmid library screening using RecA-coated biotinylated probes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9591–9595. doi: 10.1073/pnas.83.24.9591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rounds D., Brueckner M., Ward D. C. Isolation of murine telomere-proximal sequences by affinity capture and PCR. Genomics. 1995 Oct 10;29(3):616–622. doi: 10.1006/geno.1995.9958. [DOI] [PubMed] [Google Scholar]
- Sonti S. V., Griffor M. C., Sano T., Narayanswami S., Bose A., Cantor C. R., Kausch A. P. Large scale isolation of expression vector cassette by magnetic triple helix affinity capture. Nucleic Acids Res. 1995 Oct 11;23(19):3995–3996. doi: 10.1093/nar/23.19.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tagle D. A., Swaroop M., Lovett M., Collins F. S. Magnetic bead capture of expressed sequences encoded within large genomic segments. Nature. 1993 Feb 25;361(6414):751–753. doi: 10.1038/361751a0. [DOI] [PubMed] [Google Scholar]
- Wang Z., Brown D. D. A gene expression screen. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11505–11509. doi: 10.1073/pnas.88.24.11505. [DOI] [PMC free article] [PubMed] [Google Scholar]