Using Synthetic Oligonucleotides as Probes

Allan Duby1, Kenneth A. Jacobs2, Anthony Celeste2

1 The University of Texas Health Science Center at Dallas, Dallas, Texas, 2 Genetics Institute, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.4
DOI:  10.1002/0471142727.mb0604s09
Online Posting Date:  May, 2001
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Abstract

The protocols in this unit describe procedures for using mixtures of 32P‐labeled oligonucleotides to screen recombinant DNA clones bound to nitrocellulose filters. A partial amino acid sequence of a protein is used to predict the nucleotide sequence of the gene that would encode it. A mixture of oligonucleotides is chosen that includes all possible nucleotide sequences encoding that amino acid sequence. This mixture of oligonucleotides is then used to screen a recombinant DNA library for the corresponding clones. In some cases however, the exact nucleotide sequence of a desired clone is known and it is possible to use a unique oligonucleotide as a probe.

     
 
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Table of Contents

  • Basic Protocol 1: Hybridization in Sodium Chloride/Sodium Citrate (SSC)
  • Basic Protocol 2: Hybridization in Tetramethylammonium Chloride (TMAC)
  • Support Protocol 1: Labeling the 5′ Ends of Mixed Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hybridization in Sodium Chloride/Sodium Citrate (SSC)

  Materials
  • Membrane filters bearing plasmid, bacteriophage, or cosmid libraries (units 6.1 & 6.2)
  • 3× SSC/0.1% SDS
  • recipePrehybridization solution
  • recipeSSC hybridization solution
  • 6× SSC/0.05% sodium pyrophosphate, prewarmed to wash temperature
  • Filter forceps (e.g., American Scientific Products #2568‐1)
  • Sealable bags (or equivalent)
  • Additional reagents and equipment for autoradiography ( appendix 3A)

Basic Protocol 2: Hybridization in Tetramethylammonium Chloride (TMAC)

  Materials
  • Nitrocellulose or nylon membrane filters bearing plasmid, bacteriophage, or cosmid libraries (units 6.1 and 6.2)
  • 150‐mm LB agarose plates (unit 1.1), prewarmed to 37°C
  • 2× SSC/0.5% SDS/ 50 mM EDTA, pH 8.0, prewarmed to 50°C
  • recipeTMAC hybridization solution, prewarmed to hybridization temperature
  • recipeTMAC wash solution
  • 2× SSC/0.1% SDS
  • 15‐cm glass crystallizing dishes
  • Filter forceps (e.g., American Scientific Products #2568‐1)
  • Additional reagents and equipment for autoradiography ( appendix 3A)

Support Protocol 1: Labeling the 5′ Ends of Mixed Oligonucleotides

  Materials
  • 2.5 to 250 pmol mixed oligonucleotides
  • [γ‐32P]ATP (>7000 Ci/mmol)
  • 25 to 50 U T4 polynucleotide kinase (unit 3.10) and 10× kinase buffer (unit 3.4)
  • Ice‐cold 10% trichloroacetic acid (TCA)
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Figures

Videos

Literature Cited

Literature Cited
   Connor, B.J., Reyes, A.A., Morin, C., Itakura, K., Teplitz, R.L., and Wallace, R.B. 1983. Detection of sickle cell βs‐globin allele by hybridization with synthetic oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 80:278‐282.
   Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S.D., Kaufman, R.J., Mufson, A., Seehra, J., Jones, S.S., Hewick, R., Fritsch, E.F., Kawakita, M., Shimizu, T., and Miyake, T. 1985. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313:806‐810.
   Jacobs, K.A., Rudersdorf, R., Neill, S.D., Dougherty, J.P., Brown, E.L., and Fritsch, E.F. 1988. The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium salt solutions: Application to identifying recombinant DNA clones. Nucl. Acids Res. In press.
   Lathe, R. 1985. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J. Mol. Biol. 183:1‐12.
   Melchior, W.B. and von Hippel, P.H. 1973. Alteration of the relative stability of dA‐dT and dG‐dC base pairs in DNA. Proc. Natl. Acad. Sci. U.S.A. 70:298‐302.
   Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabe, S., Waterfield, M.D., and Ullrich, A. 1986. The complete primary structure of protein kinase C—the major phorbol ester receptor. Science 233:853‐859.
   Richardson, C.C. 1971. Polynucleotide kinase from Escherichia coli infected with bacteriophage T4. Nucl. Acids Res. 2:815.
   Suggs, S.V., Wallace, R.B., Hirose, T., Kawashima, E.H., and Itakura, K. 1981. Use of synthetic oligonucleotides as hybridization probes: Isolation of cloned cDNA sequences for human β2‐microglobulin. Proc. Natl. Acad. Sci. U.S.A. 78:6613.
   Wallace, R.B., Schaffer, J., Murphy, R.F., Bonner, J., Hirose, T., and Itakura, K. 1979. Hybridization of synthetic oligodeoxyribonucleotides to X174 DNA: The effect of single base pair mismatch. Nucl. Acids Res. 6:3543.
   Wetmur, J.G. 1976. Hybridization and renaturation kinetics of nucleic acids. Ann. Rev. Biophys. Bioeng. 5:337‐361.
   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. U.S.A. 82:1585‐1588.
   Woods, D.E., Miarkham, A.F., Ricker, A.T., Goldberger, G., and Colten, H.R. 1982. Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product. Proc. Natl. Acad. Sci. U.S.A. 79:5661‐5665.
   Yang, J.H., Ye, J.H., and Wallace, D.C. 1984. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening. Nucl. Acids Res. 12:837‐843.
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