Direct Selection of cDNAs Using Genomic Contigs

Michael Lovett1

1 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 6.3
DOI:  10.1002/0471142905.hg0603s00
Online Posting Date:  May, 2001
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Abstract

In the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biotinylated genomic DNAs. The selected sequences are amplified by PCR and rehybridized to create a secondary‐selected enriched cDNA library. Biotinylation of cloned genomic DNA is presented in the first support protocol. Linker addition and suppression of repeats in uncloned DNA is presented in the second support protocol and insert amplification and suppression of repeats from libraries is detailed in the third support protocol.In the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biot.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Hybridization Selection of cDNAs
  • Support Protocol 1: Biotinylation of Cloned Genomic DNA
  • Support Protocol 2: Linker Addition and Suppression of Repeats in Uncloned Starting cDNA
  • Support Protocol 3: Insert Amplification and Repeat Suppression of cDNAs from a Library
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Hybridization Selection of cDNAs

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • protocol 220 µg/ml biotinylated genomic DNA contig (YAC DNA; first protocol 2support protocol)
  • Mineral oil
  • Prehybridized cDNA (second protocol 3support protocol or third protocol 4support protocol)
  • recipe2× hybridization solution (see recipe)
  • 10 mg/ml streptavidin‐coated magnetic bead (Dynabeads M280; Dynal)
  • recipeBinding buffer (see recipe)
  • 1× SSC/0.1% (w/v) SDS ( appendix 2D), room temperature
  • 0.1× SSC/0.1% (w/v) SDS ( appendix 2D),65°C
  • 100 mM NaOH (prepare fresh)
  • 1 M Tris⋅Cl, pH 7.5
  • Sephadex G‐50 spin column (Pharmacia)
  • 10 µM PCR primers: recipeOLIGO 1 primer if using primary cDNA or recipe10F/10R primer mix if using cDNAs amplified from a λgt10 library (see reciperecipes)
  • 1% and 0.8% agarose gels (unit 2.7)
  • PrimErase column (Stratagene) or Sepharose CL‐4B column (Pharmacia)
  • Hybridization probes, radiolabeled with 32P by random priming and spin‐column purified ( appendix 3E), 100 ng for each hybridization: positive‐control reporter DNA, negative‐control reporter DNA,COT‐1 DNA (GIBCO/BRL), ribosomal control probe, and vector control DNA (e.g., pYAC4)
  • EcoRI restriction endonuclease and 10× buffer
  • recipeNA‐45 elution solution (see recipe)
  • EcoRI‐digested λgt10 DNA (Promega)
  • C600Hfl E. coli cells (CPMB Table 97.80.4711)
  • Phage dilution buffer: 20 mM Tris⋅Cl (pH 8.0)/20 mM MgCl 2 (store sterile at room temperature)
  • 5 µg human genomic DNA ( appendix 3B)
  • 5 µg genomic DNA from a somatic cell hybrid (unit 3.1) containing only the chromosomal region of interest (if available)
  • 5 µg genomic DNA from the rodent parental cell line used to derive the somatic cell hybrid
  • 100 ng total yeast DNA (strain AB1380)
  • 100 ng total DNA from each of the starting YACs comprising the contig
  • 100 ng total DNA from an unrelated YAC clone
  • 95°C heating block
  • 65°C incubator with shaker
  • Magnetic particle concentrator (Dynal)
  • NA‐45 paper (Schleicher & Schuell)
  • 96‐well microtiter plate
  • Additional reagents and equipment for spin‐column purification of labeled DNA ( appendix 3E), PCR amplification of DNA (CPMB UNIT ), agarose gel electrophoresis and Southern hybridization (unit 2.7), quantitation of nucleic acids ( appendix 3D), slot blotting (CPMB UNIT ), phenol extraction and ethanol precipitation ( appendix 3C), electroelution of DNA from agarose gels (CPMB UNIT ), construction of a cDNA library (CPMB UNIT ), and screening a recombinant phage library (CPMB UNITS , & )
CAUTION: Radiolabeled probes are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Biotinylation of Cloned Genomic DNA

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Chromosome DNA preparations from individual YAC clones, in agarose blocks (five blocks per YAC clone)
  • 1% agarose gel (unit 2.7)
  • Agarose gel suitable for contour‐clamped homogeneous electric field (CHEF) electrophoresis
  • 0.5 µg/ml ethidium bromide solution ( appendix 2D)
  • DpnII restriction endonuclease and 10× buffer
  • GeneClean II kit (Bio101)
  • TE buffer, pH 8.0
  • Nick translation kit (Nick Translation System, GIBCO/BRL)
  • 0.4 mM biotin‐16‐dUTP (Boehringer Mannheim)
  • FLASH chemiluminescence detection system (Stratagene)
  • 15°C water bath
  • Additional reagents and equipment for preparation of high‐molecular‐weight DNA in agarose blocks (unit 5.1), agarose gel electrophoresis (unit 2.7), CHEF (unit 5.1), nucleic acid quantitation ( appendix 3D),spin‐column procedure for purification of labeled DNA ( appendix 3E), and ethanol precipitation of DNA ( appendix 3C)
CAUTION: Ethidium bromide is hazardous; see appendix 2A for guideline on handling, storage, and disposal.

Support Protocol 2: Linker Addition and Suppression of Repeats in Uncloned Starting cDNA

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 3 µg random‐primed, blunt‐ended,double‐stranded cDNA in 22 µl water, average length between 500 bp and 1 kb (CPMB UNIT )
  • 10× T4 DNA ligase buffer ( appendix 2D)
  • recipe1 µg/µl phosphorylated cDNA linkers (see recipe)
  • 1 U/µl T4 DNA ligase (Boehringer Mannheim)
  • PrimErase column (Stratagene) or Sepharase CL‐4B column (Pharmacia)
  • Sephadex G‐50 spin column (Pharmacia)
  • 2 µg human COT‐1 DNA (GIBCO/BRL)
  • 100 ng ribosomal DNA
  • 10 ng pYAC4 vector DNA (Sigma) digested with DpnII or 10 ng digested vector DNA for cosmid or phage selections
  • Mineral oil
  • recipe2× hybridization solution (see recipe)
  • 95°C heating block
  • 65°C water bath or oven
  • Additional equipment and reagents for phenol extraction and ethanol precipitation of DNA ( appendix 3C) and spin‐column procedure for purification of labeled DNA ( appendix 3E)
NOTE: Ribosomal‐blocking DNA is only necessary for selections with YACs and can be dispensed with for cosmid or phage selections. It can consist of 1 µg total yeast DNA from the strain AB1380, 100 ng cloned ribosomal genes (including the 5.8S rRNA), or preferably 100 ng cDNA products from a cDNA synthesis conducted on poly(A) RNA [the oligo(dT)‐cellulose flowthrough; see CPMB UNIT ].

Support Protocol 3: Insert Amplification and Repeat Suppression of cDNAs from a Library

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Starting cDNA library cloned in λgt10 (≥106 pfu/µl; other vector systems can be substituted but the vector primers must changed as well)
  • 3 U/ml Hot Tub DNA polymerase and 10× Hot Tub buffer (Amersham)
  • 2.5 mM 4dNTP mix ( appendix 2D)
  • recipe10× 10F/10R PCR primer mix (see recipe)
  • Mineral oil
  • 1% agarose gel (unit 2.7)
  • 1 µg EcoRI‐digested phage DNA from a bulk preparation of the entire starting cDNA library (CPMB UNIT )
  • Molecular size markers
  • PrimErase column (Stratagene) or Sepharose CL‐4B column (Pharmacia)
  • 2 µg human COT‐1 DNA (GIBCO/BRL)
  • 100 ng ribosomal DNA
  • 10 ng phage or plasmid vector DNA
  • recipe2× hybridization solution (see recipe)
  • Additional reagents and equipment for PCR amplification of DNA (CPMB UNIT ),agarose gel electrophoresis (unit 2.7), phenol extraction and ethanol precipitation of DNA ( appendix 3C), and spin‐column procedure for purification of labeled DNA ( appendix 3E)
NOTE: Ribosomal‐blocking DNA is only necessary for selections with YACs and can be dispensed with for cosmid or phage selections. It can consist of 1 µg total yeast DNA from the strain AB1380, 100 ng cloned ribosomal genes (including the 5.8S rRNA), or preferably 100 ng cDNA products from a cDNA synthesis conducted on poly(A) RNAs [the oligo(dT)‐cellulose flowthrough; see CPMB UNIT
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Figures

Videos

Literature Cited

Literature Cited
   Elvin, P.., Slynn, G., Black, D., Graham, A., Butler, R., Riley, J., Anand, R. and, Markham, A.F. 1990. Isolation of cDNA clones using yeast artificial chromosome probes. Nucl Acids Res. 18:3913‐3917.
   Innis, M.A. and Gelfand, D.H. 1990. Optimization of PCRs. In PCR Protocols: A Guide to Methods and Applications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.) pp 1‐12. Academic Press, N.Y.
   Korn, B., Sedlacek, Z., Manca, A., Kioschis, P., Konecki, D., Lehrach, H. and, Poustka, A. 1992. A strategy for the selection of transcribed sequences in the Xq28 region. Hum. Mol. Genet. 1:235‐242.
  Lovett, M., Kere, J. and, Hinton, L.M. 1991. Direct selection: A method for the isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. U.S.A. 88:9628‐9632.
   Morgan, J.G., Dolganov, G.M., Robbins, S.E., Hinton, L.M. and, Lovett, M. 1992. The selective isolation of novel cDNAs encoded by the regions surrounding the human interleukin 4 and 5 genes. Nucl. Acids Res. 20:5173‐5179.
   Parimoo, S., Patanjali, S.R., Shukla, H., Chaplin, D.D. and, Weissman, S.M. 1991. cDNA selection: Efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc. Natl. Acad. Sci. U.S.A. 88:9623‐9627.
   Reyes, G.R., Bradley, D.W. and, Lovett, M. 1992. New strategies for the isolation of low abundance viral and host cDNAs: Application to cloning of the Hepatitis E virus and analysis of tissue‐specific transcription. Semi. Liver Dis. 12:289‐300.
   Wallace, M.R., Marchuk, D.A., Anderson, L.B., Letcher, R., Odeh, H.M., Saulino, A.M., Fountain, J.W., Brerton, A., Nicholson, J., Mitchell, A.L., Brownstein, B.H. and, Collins, F.S. 1990. Type 1 neurofibromatosis gene:Identification of a large transcript disrupted in three NF1 patients. Science 249:181‐186.
   Young, B.D. and Anderson, M.L.M. 1987. Quantitative analysis of solution hybridization. In Nucleic acid hybridization,a practical approach. (B.D. Hames and S.J. Higgins, eds.) pp. 47‐71. IRL Press, Oxford.
Key Reference
   Morgan et al., 1992. See above.
  Describes direct selection in solution, biotinylation, and the use of primary and uncloned cDNAs.
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