Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells

  • Csilla Csortos
  • Virginie Lazar
  • Joe G. N. Garcia
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 30)


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.


cDNA Library Hybridization Buffer Myosin Light Chain Kinase Library Screening Nucleic Acid Probe 
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  1. 1.
    Benton, W. D. and Davis, R. W. (1977) Screening lgt recombinant clones by hybridisation to single plaques in situ. Science 196, 180–182.CrossRefPubMedGoogle Scholar
  2. 2.
    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.Google Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    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.Google Scholar
  5. 5.
    Denhardt, D. (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys, Res. Commun. 23, 641–646.CrossRefGoogle Scholar
  6. 6.
    Southern, E. M. (1975) Detection of specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefPubMedGoogle Scholar
  8. 8.
    Lathe, R. (1985) Synthetic oligonucleotide probes deduced from amino acid sequence data. J. Mol. Biol. 183, 1–12.CrossRefPubMedGoogle Scholar
  9. 9.
    Skalka, A. and Shapire, L. (1976) In situ immunoassays for gene translation produces in phage plaques and bacterial colonies. Gene 1, 65.CrossRefPubMedGoogle Scholar
  10. 10.
    Young, R. A. and Davis, R. W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80, 1194–1198.CrossRefPubMedGoogle Scholar
  11. 11.
    Benson, S. and Taylor, R. K. (1984) A rapid small-scale procedure for isolation of phage lambda DNA. Biotechniques 2, 126,127.Google Scholar
  12. 12.
    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, 1401–1406CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Csilla Csortos
    • 1
  • Virginie Lazar
    • 1
  • Joe G. N. Garcia
    • 1
  1. 1.Department of MedicineIndiana Univeristy School of MedicineIndianapolis

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