Abstract
Libraries of cDNA clones are valuable resources for analyzing the expression, structure and regulation of genes, and for studying protein functions and interactions. Full-length cDNA clones provide information about intron and exon structures, splice junctions, and 5′ and 3′ untranslated regions (UTRs). Open reading frames (ORFs) derived from cDNA clones can be used to generate constructs allowing the expression of both wild-type proteins and proteins tagged at their amino or carboxy terminus. Thus, obtaining full-length cDNA clones and sequences for most or all genes in an organism is essential for understanding genome functions. EST sequencing samples cDNA libraries at random, an approach that is most useful at the beginning of large-scale screening projects. As projects progress towards completion, however, the probability of identifying unique cDNAs by EST sequencing diminishes, resulting in poor recovery of rare transcripts. Here we describe an adapted, high-throughput protocol intended for the recovery of specific, full-length clones from plasmid cDNA libraries in 5 d.
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References
Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning: A Laboratory Manual Vol. 1, edn. 2, 1.90–1.110, 6.4–6.13 (Cold Spring Harbor Laboratory Press, Plainview, NY, 1989).
Adams, M.A. et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–1656 (1991).
McCombie, W.R. et al. Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues. Nat. Genet. 1, 124–131 (1992).
Delseny, M., Cooke, R., Raynal, M. & Grellet, F. The Arabidopsis thaliana cDNA sequencing projects. FEBS Lett. 405, 129–132 (1997).
Rubin, G.M. et al. A Drosophila complementary DNA resource. Science 287, 2222–2224 (2000).
Mocharla, H., Mocharla, R. & Hodes, M.E. Coupled reverse transcription–polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping. Gene 93, 271–275 (1990).
Munroe, D.J. et al. Systematic screening of an arrayed cDNA library by PCR. Proc. Natl. Acad. Sci. USA 92, 2209–2213 (1995).
Frohman, M.A., Dush, M.K. & Martin, G.R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002 (1988).
Huang, S.H., Chen, S.H. & Jong, A.Y. Use of inverse PCR to clone cDNA ends. Methods Mol. Biol. 221, 51–58 (2003).
Ochman, H., Gerber, A.S. & Hartl, D.L. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623 (1988).
Triglia, T., Peterson, M.G. & Kemp, D.J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16, 8186 (1988).
Green, I.R. & Sargan, D.R. Sequence of the cDNA encoding ovine tumor necrosis factor-α: problems with cloning by inverse PCR. Gene 109, 203–210 (1991).
Haerry, T.E. & O'Connor, M.B. Isolation of Drosophila activin and follistatin cDNAs using novel MACH amplification protocols. Gene 291, 85–93 (2002).
Hoskins, R.A. et al. Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP). Nucleic Acids Res. 33, e185–115 (2005).
Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. & Mattick, J.S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 (1991).
Worthington, M.T., Luo, R.Q. & Pelo, J. Copacabana method for spreading E. coli and yeast colonies. BioTechniques 30, 738–740, 742 (2001).
Acknowledgements
We thank S. Park, J. Karpen and members of the Berkeley Drosophila Genome Project for technical support; and G. Karpen for critically reading the manuscript. This work was supported by a grant from the National Institutes of Health (HG002673 to S.E.C.) through the U.S. Department of Energy (contract number DE AC02 05CH11231).
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R.A.H. conceived the technique and performed the proof-of-concept experiments. M.S., C.Y. and K.H.W. adapted the technique for high-throughput screening with input from R.A.H, R.A.G and S.E.C. R.A.G and R.S. wrote custom software scripts to design PCR primers. J.W.C., M.S. and S.E.C. chose targets based on gene predictions. S.E.C. and J.W.C. analyzed data resulting from the screens. C.Y. and K.H.W. wrote the high-throughput protocol, with guidance from R.A.H. and M.S. S.E.C. edited the protocol for publication and provided general supervision.
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Wan, K., Yu, C., George, R. et al. High-throughput plasmid cDNA library screening. Nat Protoc 1, 624–632 (2006). https://doi.org/10.1038/nprot.2006.90
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DOI: https://doi.org/10.1038/nprot.2006.90
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