Abstract
In the study of biological systems, often the amount of starting material available for molecular analysis is limiting. In some situations, only a few cells in a tissue are expressing genes of interest or the tissue is in limited supply. A cDNA library from the targeted cells is preferable to a library constructed from the entire tissue containing these cells, because in the latter case, genes expressed in the targeted cells may be too rare to be detected. cDNA clones of genes expressed in small amounts of material are often hard to obtain because the construction of conventional cDNA libraries requires microgram amounts of poly A+ RNA (1).
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References
Gasser, C. S., Budelier, K. A., Smith, A. G., Shah, D. M., and Fraley, R.T. (1989) Isolation of tissue-specific cDNAs from tomato pistils. Plant Cell 1, 15–24.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Arnheim, N., Li, H., and Cui, X. (1990) PCR analysis of DNA sequences in single cells: single sperm gene mapping and genetic disease diagnosis. Genomics 8, 415–419.
Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) 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.
Ohara, O., Dorit R. L., and Gilbert, W. (1989) One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. USA 86, 5673–5677.
Akowitz, A. and Manuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81, 295–306.
Welsh, J., Liu, J.-P., and Efstratiadis, A. (1990) Cloning of PCR-amplified total cDNA: construction of a mouse oocyte library. Genet. Anal. Technol. Appl. 7, 5–17.
Domec, C., Garbay, B., Founier, M., and Bonnet, J. (1990) cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification. Anal. Biochem. 188, 422–426.
Jepson, I., Bray, J., Jenkins, G., Schuch, W., and Edwards, K. (1991) A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue. Plant Mol. Biol. Reporter 9, 131–138.
Gurr, S. J., McPherson, M. J., Scollan, C., Atkinson, H. J., and Bowles, D. J. (1991) Gene expression in nematode-infected plant roots. Mol. Gen. Genet. 226, 361–366.
Ko, M. S. H., Ko, S. B. H., Takahashi, N., Nishiguchi, K., and Abe, K. (1990) Unbiased amplification of a highly complex mixture of DNA fragments by “lone linker”-tagged PCR. Nucleic Acids Res. 18, 4293,4294.
Froussard, P. (1992) A random-PCR method (rPCR) to construct whole cDNA library from low amounts of RNA. Nucleic Acids Res. 20, 2900.
Jakobsen, K. S., Breivold, E., and Homes, E. (1990) Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres. Nucleic Acids Res. 18, 3669.
Raineri, I., Moroni, C., and Senn, H. P. (1991) Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Res. 19, 4010.
Lambert, K. N. and Williamson, V. M. (1993) cDNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Res. 21, 775,776.
Kaufman, D. L. and Evans, G. A. (1990) Restriction endonuclease cleavage at the termini of PCR products. BioTechniques 9, 304–306.
Clark, J. M. (1988) Novel non-template nucleotide addition reactions catalysed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 20, 9677–9686.
Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269.
Fernandez, J. M., Mc Atee, C., and Herrnstadt, C. (1990) Advances in cDNA technology. Am. Biotechnol. Lab. 8, 46,47.
Stoker, A. W. (1990) Cloning of PCR products after defined cohesive terminal are created with T4 DNA polymerase. Nucleic Acids Res. 18, 4290.
Kripamoy, A., Kusano, T., Suzuki, N., and Kitagawa, Y. (1990) An Improved method for the construction of high efficiency cDNA library in plasmid or lambda vector. Nucleic Acids Res. 18, 1071.
Hung, T., Mak, K., and Fong, K. (1990) A specificity enhancer for polymerase chain reaction. Nucleic Acids Res. 18, 4953.
Wood, W. I., Gitschier, J., Lasky, L. A., and Lawn, R. M. (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc. Natl. Acad. Sci. USA 82, 1585–1588.
Karrer, E. E., Lincoln, J. E., Hogenhout, S., Bennett, A. B., Bostock, R. M., Martineau, B., Lucas, W. J., Gilchrist, D. G., and Alexander, D. (1995) In situ isolation of mRNA from individual plant cells creation of cell-specific cDNA libraries. Proc. Natl. Acad. Sci. USA 92, 3814–3818.
Rochester, D. E., Winer, J. A., and Shah, D. M. (1986) The structure and expression of maize genes encoding the major heat shock protein, hsp70. EMBO J. 5, 451–458.
Haymerie, H., Herz, J., Bressan, G. M., Frank, R., and Stanley, K. K. (1986) Efficient construction of cDNA libraries in plasmid expression vectors using an adapter strategy. Nucleic Acids Res. 14, 8615–8624.
D’Souza, C. R., Deugau, K. V., and Spencer, J. H. (1989) A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors. Biochem. Cell Biol. 61, 205–209.
Bhat, G. J., Lodes, M. J., Myler, P. J., and Stuart, K. D. (1990) A simple method for cloning blunt ended DNA fragments. Nucleic Acids Res. 19, 398.
Challberg, M. D. and Englund, P. T. (1980) Specific labeling of 3′ termini with T4 DNA polymerase, in Methods in Enzymology, vol. 65, Nucleic Acids, Part I (Grossman, L. and Moldave, K., eds.), Academic, New York, pp. 39–43.
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Lambert, K.N., Williamson, V.M. (1997). cDNA Library Construction from Small Amounts of RNA Using Paramagnetic Beads and PCR. In: Cowell, I.G., Austin, C.A. (eds) cDNA Library Protocols. Methods in Molecular Biology™, vol 69. Humana Press. https://doi.org/10.1385/0-89603-383-X:1
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DOI: https://doi.org/10.1385/0-89603-383-X:1
Publisher Name: Humana Press
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