Abstract
Complementary DNA (cDNA) sequencing can be used to sample an organism’s transcriptome, and the generated EST sequences can be used for a variety of purposes. They are especially important for enhancing the utility of a genome sequence or for providing a gene catalog for a genome that has not or will not be sequenced. In planning and executing a cDNA project, several criteria must be considered. One should clearly define the project purpose, including organism tissue(s) choice, whether those tissues should be pooled, ability to acquire adequate amounts of clean and well-preserved tissue, choice of type(s) of library, and construction of a library (or libraries) that is compatible with project goals. In addition, one must possess the skills to construct the library (or libraries), keeping in mind the number of clones that will be necessary to meet the project requirements. If one is inexperienced in cDNA library construction, it might be wise to outsource the library production and/or sequence and analysis to a sequencing center or to a company that specializes in those activities. One should also be aware that new sequencing platforms are being marketed that may offer simpler protocols that can produce cDNA data in a more rapid and economical manner. Of course, the bioinformatics tools will have to be in place to de-convolute and aid in data analysis for these newer technologies. Possible funding sources for these projects include well-justified grant proposals, private funding, and/or collaborators with available funds.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mitreva, M., McCarter, J. P., Martin, J., Dante, M., Wylie, T., Chiapelli, B., Pape, D., Clifton, S. W., Nutman, T. B., and Waterston, R. H. (2004) Comparative genomics of gene expression in the parasitic and free-living nematodes Strongyloides stercoralis and Caenorhabditis elegans. Genome Res 14, 209–20.
Che, S., and Ginsberg, S. D. (2004) Amplification of RNA transcripts using terminal continuation. Lab Invest 84, 131–7.
Ranjit, N., Jones, M. K., Stenzel, D. J., Gasser, R. B., and Loukas, A. (2006) A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy. Inter J Parasitol 36, 701–10.
Whitfield, C. W., Band, M. R., Bonaldo, M. F., Kumar, C. G., Liu, L., Pardinas, J. R., Robertson, H. M., Soares, M. B., and Robinson, G. E. (2002) Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee. Genome Res. 12, 555–66.
Reymond, A., Marigo, V., Yaylaoglu, M. B., Leoni, A., Ucla, C., Scamuffa, N., Caccioppoli, C., Dermitzakis, E. T., Lyle, R., Banfi, S., Eichele, G., Antonarakis, S. E., and Ballabio, A. (2002) Human chromosome 21 gene expression atlas in the mouse. Nature 420, 582–86.
Reeves, R. H. (2002) Functional genomics: A time and place for every gene. Nature 420, 518–19.
Mitreva, M., Appleton, J., McCarter, J. P., and Jasmer, D. P. (2005) Expressed sequence tags from life cycle stages of Trichinella spiralis: Application to biology and parasite control. Vet Parasitol 132, 13–17.
Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S. A. (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucl Acids Res 25, 3957–58.
Kitade, Y., Ootsuka, S., Iitsuka, O., and Saga, N. (2003) Effect of DMSO on PCR of Porphyra yezoensis (Rhodophyta) gene. J Appl Phycol 15, 555–57.
Ohara, O., and Temple, G. (2001) Directional cDNA library construction assisted by the in vitro recombination reaction. Nucl Acids Res 29, e22.
Landy, A. (1989) Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Ann Rev Biochem 58, 913–41.
Abremski, K., and Hoess, R. (1984) Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. J Biol Chem 259, 1509–14.
Maruyama, K., and Sugano, S. (1994) Oligo-capping: A simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171–4.
Suzuki, Y., and Sugano, S. (2001) Construction of full-length-enriched cDNA libraries. The oligo-capping method. Methods Mol Biol 175, 143–53.
Carninci, P., Kvam, C., Kitamura, A., Ohsumi, T., Okazaki, Y., Itoh, M., Kamiya, M., Shibata, K., Sasaki, N., Izawa, M., Muramatsu, M., Hayashizaki, Y., and Schneider, C. (1996) High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–36.
Carninci, P., and Hayashizaki, Y. (1999) High-efficiency full-length cDNA cloning. Methods Enzymol 303, 19–44.
Vandenberghe, A. E., Meedel, T. H., and Hastings, K. E. M. (2001) mRNA 5'-leader trans-splicing in the chordates. Genes Dev 15, 294–303.
Davis, R. E. (1997) Surprising diversity and distribution of spliced leader RNAs in flatworms. Mol Biochem Parasitol 87, 29–48.
Davis, R. E., Singh, H., Botka, C., Hardwick, C., Ashraf el Meanawy, M., and Villanueva, J. (1994) RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs. J Biol Chem 269, 20026–30.
Nilsen, T. W. (1993) Trnas-splicing of nematode premessenger RNA. Ann Rev Microbiol 47, 413–40.
Ebel, C., Frantz, C., Paulus, F., and Imbault, P. (1999) Trans-splicing and cis-splicing in the colourless Euglenoid, Entosiphon sulcatum. Curr Genet 35, 542–50.
Frantz, C., Ebel, C., Paulus, F., and Imbault, P. (2000) Characterization of trans-splicing in Euglenoids. Curr Genet 37, 349–55.
Tessier, L. H., Keller, M., Chan, R. L., Fournier, R., Weil, J. H., and Imbault, P. (1991) Short leader sequences may be transferred from small RNAs to pre-mature mRNAs by trans-splicing in Euglena. Embo J 10, 2621–5.
Krause, M., and Hirsh, D. (1987) A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49, 753–61.
Evans, D., and Blumenthal, T. (2000) trans Splicing of Polycistronic Caenorhabditis elegans Pre-mRNAs: Analysis of the SL2 RNA. Mol Cell Biol 20, 6659–67.
Stover, N. A., and Steele, R. E. (2001) Trans-spliced leader addition to mRNAs in a cnidarian. Proc Natl Acad Sci USA 98, 5693–98.
Wylie, T., Martin, J., Dante, M., Mitreva, M., Clifton, S. W., Chinwalla, A., Waterston, R. H., Wilson, R. K., and McCarter, J. P. (2004) Nematode.net: A tool for navigating sequences from parasitic and free-living nematodes. Nucleic Acids Res 32, D423–D26.
Soares, M. B., Bonaldo, M. F., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A. (1994) Construction and characterization of a normalized cDNA library. Proc Natl Acad Sci USA 91, 9228–32.
Ciavatta, V., and Cairney, J. (2000) Isolation of full-length cDNA clones using SMART cDNA and a biotin-streptavidin bead system. Biotechniques 29, 444–6, 48, 50.
Hara, E., Kato, T., Nakada, S., Nakajima, T., Tsurui, H., and Oda, K. (1990) Construction of a subtracted cDNA library using oligo(dT)-latex. Nucleic Acids Symp Ser 22, 29–30.
Zhulidov, P. A., Bogdanova, E. A., Shcheglov, A. S., Shagina, I. A., Vagner, L. L., Khazpekov, G. L., Kozhemiako, V. V., Luk'ianov, S. A., and Shagin, D. A. (2005) A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Bioorg Khim 31, 186–94.
Zhulidov, P. A., Bogdanova, E. A., Shcheglov, A. S., Vagner, L. L., Khaspekov, G. L., Kozhemyako, V. B., Matz, M. V., Meleshkevitch, E., Moroz, L. L., Lukyanov, S. A., and Shagin, D. A. (2004) Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res 32, e37.
Sargent, T. D., and Dawid, I. B. (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135–9.
Duguid, J. R., and Dinauer, M. C. (1990) Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucl Acids Res 18, 2789–92.
Duguid, J. R., Rohwer, R. G., and Seed, B. (1988) Isolation of cDNAs of Scrapie-Modulated RNAs by Subtractive Hybridization of a cDNA Library. Proc Natl Acad Sci 85, 5738–42.
Rubenstein, J. L. R., Brice, A. E. J., Ciaranello, R. D., Denney, D., Porteus, M. H., and Usdin, T. B. (1990) Subtractive hybridization system using single-stranded phagemids with directional inserts. Nucl Acids Res 18, 4833–42.
Rodriguez, I. R., and Chader, G. J. (1992) A novel method for the isolation of tissue-specific genes. Nucl Acids Res 20, 3528.
Sharma, P., Lanneborg, A., and Stougaard, P. (1993) PCR-based construction of subtractive cDNA library using magnetic beads. Biotechniques 15, 610–12.
Lopez-Fernandez, L. A., and del Mazo, J. (1993) Construction of subtractive cDNA libraries from limited amounts of mRNA and multiple cycles of subtraction. Biotechniques 15, 658–9.
Li, W. B., Gruber, C. E., Lin, J. J., Lim, R., D'Alessio, J. M., and Jessee, J. A. (1994) The isolation of differentially expressed genes in fibroblast growth factor stimulated BC3H1 cells by subtractive hybridization. Biotechniques 16, 722–9.
Schoen, T. J., Mazuruk, K., Chader, G. J., and Rodriguez, I. R. (1995) Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive cloning technique. Biochem Biophys Res Commun 213, 181–88.
Leygue, E. R., Watson, P. H., and Murphy, L. C. (1996) Identification of differentially expressed genes using minute amounts of RNA. Biotechniques 21, 1008–10, 12.
Blumberg, B., and Belmonte, J. C. (1999) Subtractive hybridization and construction of cDNA libraries. Methods Mol Biol 97, 555–74.
Forster, A. C., McInnes, J. L., Skingle, D. C., and Symons, R. H. (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucl Acids Res 13, 745–61.
Sive, H. L., and John, T. S. (1988) A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucl Acids Res 16, 10937.
McInnes, J. L., Forster, A. C., Skingle, D. C., and Symons, R. H. (1990) Preparation and uses of photobiotin. Methods Enzymol 184, 588–600.
Wylie, T., Martin, J. C., Dante, M., Mitreva, M. D., Clifton, S. W., Chinwalla, A., Waterston, R. H., Wilson, R. K., and McCarter, J. P. (2004) Nematode.net: A tool for navigating sequences from parasitic and free-living nematodes. Nucl Acids Res 32, D423–6.
Mitreva, M., Blaxter, M. L., Bird, D. M., McCarter, J. P. (2005) Comparative genomics of nematodes. Trends Genet 21, 573–81.
Acknowledgments
This work was supported by the National Human Genome Research Institute (Grant U54 HG003079).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Clifton, S.W., Mitreva, M. (2009). Strategies for Undertaking Expressed Sequence Tag (EST) Projects. In: Parkinson, J. (eds) Expressed Sequence Tags (ESTs). Methods in Molecular Biology, vol 533. Humana Press. https://doi.org/10.1007/978-1-60327-136-3_2
Download citation
DOI: https://doi.org/10.1007/978-1-60327-136-3_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-759-4
Online ISBN: 978-1-60327-136-3
eBook Packages: Springer Protocols