Abstract
Various methods are available for the construction of essentially complete cDNA or genomic libraries from many sources. Since such libraries contain thousands of members, a major difficulty is the identification of clones of interest. Final identifications are usually made by sequencing the DNA, by using the DNA to select a specific mRNA for in vitro translation, or both. Such assays are not usually suited for screening large libraries for individual clones and steps are taken to enrich for the clones of interest. The most commonly used procedures rely on cDNA hybridization probes made from an mRNA preparation that contains high levels of the sequence of interest. Variations of this general approach include the physical fractionation of the mRNA by sedimentation, gel electrophoresis, or immunoprecipitation (or absorption) of polysomes. Alternatively, if part or all of the amino acid sequence of a protein is known, it is possible to synthesize specific oligonucleotides for use either as hybridization probes themselves or as primers for the enzymatic synthesis of cDNA.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Helfman, D.M., Feramisco, J.R., Fiddes, J.C. Thomas, G.P. and Hughes, S.H. (1983) Proc Nat. Acad. Sci. U.S.A. 80, 31–35.
Goodman, H.M. (1979) Nature (London) 281, 351–355.
Kurtz, D.T. and Nicodemus, C.F. (1981) Gene 13, 145–152.
Payvar, F. and Schimke, R.T. (1979) J. Biol. Chem. 254, 7636–7642.
Vieira, J. and Messing, J. (1982) Gene 19, 259–268.
Helfman, D.M., Feramisco, J.R., Ricci, W.M. and Hughes, S.H. (1984) J. Biol. Chem. 259, 14136–14143.
Hanahan, D. (1983) J. Mol. Biol. 166, 557–580.
Hanahan, D. and Meselson, M. (1980) Gene 10, 63–67.
Helfman, D.M., Yamawaki-Kataoka, Y., Ricci, W.R., Feramisco, J.R. and Hughes, S.H. (1984) in Molecular Biology of the Cytoskeleton (Borisy, G.G, Cleveland, D.W. and Murphy, D.B., eds.) pp. 479–491, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Laemmli, U.K. (1970) Nature 227, 680–685.
Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 4350–4354.
Blattler, D.P., Garner, F., Van Slyke, K. and Bradley, A. (1972) J. Chromatogr. 64, 147–155.
Mayo, K.E., Vale, W., Rivier, J., Rosenfeld, M.G. and Evans, R.M. (1983) Nature 306, 86–88.
Lin, C.R., Chen, W.S., Kruiger, W., Stolarsky, L.S., Weber, W., Evans, R.M., Verma, I.M., Gill, G.N. and Rosenfeld, M.G. (1984) Science 224, 843–848.
Lewis, S.A., Balcarek, J.M., Krek, V., Shelanski, M. and Cowan, N.J. (1984) Proc. N t. Acad. Sci. U.S.A. 81, 2743–2746.
Brown, A.J.P., Leibold, E.A. and Munro, H.N. (1983) Proc. Nat. Acad. Sci. U.S.A. 80, 1265–1269.
Meyer, T.F., Mlawer, N. and So, M. (1982) Cell 30, 45–52.
Ohta, N., Chen, L.S. and Newton, A. (1982) Proc. Nat. Acad. Sci. U.S.A. 79, 4863–4867.
Schwarzbauer, J.E., Tamkun, J.W., Lemischka, I. and Hynes, R.O. (1983) Cell 35, 421–431.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1985 Plenum Press, New York
About this chapter
Cite this chapter
Helfman, D.M., Feramisco, J.R., Fiddes, J.C., Thomas, G.P., Hughes, S.H. (1985). Identification and Isolation of Clones by Immunological Screening of cDNA Expression Libraries. In: Setlow, J.K., Hollaender, A. (eds) Genetic Engineering: Principles and Methods. Genetic Engineering: Principles and Methods, vol 7. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-4973-0_9
Download citation
DOI: https://doi.org/10.1007/978-1-4684-4973-0_9
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4684-4975-4
Online ISBN: 978-1-4684-4973-0
eBook Packages: Springer Book Archive