Abstract
The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Benton, W. D. and Davis, R. W. (1977) Screening lgt recombinant clones by hybridisation to single plaques in situ. Science 196, 180–182.
Huynh, T. V., Young, R. A., and Davis, R. V. (1984) DNA Cloning: A Practical Approach, Vol. 1 (D.M. Glover, ed.) pp. 49–78.
Garcia, J. G. N., Lazar, V., Gilbert-McClain, L. I., Gallagher, P. J., and Verin, A. D. (1997) Myosin light chain kinase in endothelium: molecular cloning and regulation. Am. J. Respir. Cell Mol. Biol. 16, 489–494.
Williams, B. G. and Blasstner, F. R. (1980) Bacteriophage lambda vectors for DNA cloning, in Genetic Engineering Vol. 2 (J. K. Setlow and A. Mullander, eds.) p. 201.
Denhardt, D. (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys, Res. Commun. 23, 641–646.
Southern, E. M. (1975) Detection of specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.
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.
Lathe, R. (1985) Synthetic oligonucleotide probes deduced from amino acid sequence data. J. Mol. Biol. 183, 1–12.
Skalka, A. and Shapire, L. (1976) In situ immunoassays for gene translation produces in phage plaques and bacterial colonies. Gene 1, 65.
Young, R. A. and Davis, R. W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80, 1194–1198.
Benson, S. and Taylor, R. K. (1984) A rapid small-scale procedure for isolation of phage lambda DNA. Biotechniques 2, 126,127.
Ginsburg, D., Handin, R. I., Bonthron, D. T., Donlon, T. A., Bruns, G. A., Latt, S. A., and Orkin, S. H. (1985) Human von Willebrand factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localisation. Science 228, 1401–1406
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Csortos, C., Lazar, V., Garcia, J.G.N. (1999). Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells. In: Baker, A.H. (eds) Vascular Disease. Methods in Molecular Medicine™, vol 30. Humana Press. https://doi.org/10.1385/1-59259-247-3:59
Download citation
DOI: https://doi.org/10.1385/1-59259-247-3:59
Publisher Name: Humana Press
Print ISBN: 978-0-89603-731-1
Online ISBN: 978-1-59259-247-0
eBook Packages: Springer Protocols