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cDNA Library Construction Using Streptavidin-Paramagnetic Beads and PCR

  • Kris N. Lambert
  • Valerie M. Williamson
Protocol
  • 140 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

cDNA clones of genes expressed in small amounts of material can be hard to obtain because the construction of conventional cDNA libraries requires microgram amounts of poly (A)+ RNA (1). The polymerase chain reaction (PCR), which is commonly used to amplify tiny amounts of DNA (2,3), has been adapted to facilitate the construction of cDNA libraries from small quantities of poly (A)+ RNA (4, 5, 6, 7). Most of these cDNA amplification methods require multiple purification or precipitation steps to remove primers and change buffers. These steps result in significant loss of material and compromise the quality of the final library. The method presented here eliminates precipitation and chromatography steps so that all cDNA synthesis and modification reactions are conducted in a single tube.

Keywords

cDNA Library Total Nucleic Acid Paramagnetic Bead Oligonucleotide Adapter Polymerase Chain Reaction Reaction Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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. The Plant Cell 1, 15–24.PubMedGoogle Scholar
  2. 2.
    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.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    Akowitz, A. and Manuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81, 295–306.PubMedCrossRefGoogle Scholar
  5. 5.
    Welsh, J., Liu, J.-P., and Efstratiadis, A. (1990) Cloning of PCR-amplified total cDNA: construction of a mouse oocyte library. Genet. Anal. Techn. Appl. 7, 5–17.CrossRefGoogle Scholar
  6. 6.
    Domec, C., Garbay, B., Fournier, 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.PubMedCrossRefGoogle Scholar
  7. 7.
    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. Rep. 9, 131–138.CrossRefGoogle Scholar
  8. 8.
    Rosok, O., Odeberg, J., Rode, M., Stokke, T., Funderud, S., Smeland, E., and Lundeberg, J. (1996) Solid-phase method for differential display of genes expressed in hematopoietic stem cells. Biotechniques 21, 114–121.PubMedGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269.PubMedCrossRefGoogle Scholar
  11. 11.
    Fernandez, J. M., Mc Atee, C., and Herrnstadt, C. (1990) Advances in cDNA technology. American Biotechnol. Lab. 8, 46–47.Google Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Kris N. Lambert
    • 1
  • Valerie M. Williamson
    • 1
  1. 1.Department of HematologyUniversity of CaliforniaDavis

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