Identifying Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries

Ute Hochgeschwender1, Miles B. Brennan1

1 National Institute of Mental Health, Bethesda, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 6.2
DOI:  10.1002/0471142905.hg0602s00
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for identifying bacteriophage or cosmid clones that contain sequences present in the mRNA of a cell line or tissue. In the , a radiolabeled cDNA probe is synthesized by reverse transcription of poly(A)+ RNA isolated from the cell line or tissue of interest. The probe is incubated with DNA‐cellulose to remove highly repetitive sequences before it is used for hybridization analysis of filters from a phage or cosmid genomic library. After identification of positive clones from the library screen, the same probe can be used to screen Southern blots of restriction enzyme‐digested DNA from the positive clones. Support protocols describe preparation and testing of DNA‐cellulose.

     
 
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Table of Contents

  • Basic Protocol 1: Identification of Transcribed Sequences by Direct Screening with cDNA
  • Support Protocol 1: Removal of Highly Repetitive Sequences from DNA Probes Using DNA‐Cellulose
  • Support Protocol 2: Preparation of DNA‐Cellulose
  • Support Protocol 3: Testing DNA‐Cellulose
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of Transcribed Sequences by Direct Screening with cDNA

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 2 to 5 µg poly(A)+ RNA (CPMB UNITS & )
  • recipe5× reverse transcriptase (RT) buffer (see recipe)
  • 0.6 M 2‐ME
  • 1 mg/ml 12‐ to 18‐mer oligodeoxythymidylic acid [oligo(dT) 12‐18]
  • 100 mM 4dNTP mix ( appendix 2D)
  • 10 µCi/µl [α‐32P]dATP (3000 Ci/mmol)
  • 25 U/µl AMV reverse transcriptase
  • TE buffer, pH 7.0
  • 5 M NaCl
  • 100% ethanol
  • recipe1.2% alkaline agarose gel (see recipe)
  • recipeAlkaline running buffer (see recipe)
  • recipeAlkaline loading buffer (see recipe)
  • 3′‐end‐labeled, HindIII‐digested λ molecular size markers
  • Sephadex G‐50 resin
  • Nylon or nitrocellulose membrane filters bearing phage or cosmid genomic library or Southern blots of restriction enzyme–digested cloned DNA.
  • recipeHybridization solution (see recipe)
  • 2× SSC/0.1% (w/v) SDS ( appendix 2D)
  • 2.4‐cm Whatman GF/C filter discs
  • Whatman 3MM filter paper
  • Sealable bags
  • 68°C water bath
  • Additional reagents and equipment for measuring radioactivity in DNA by acid precipitation ( appendix 3E), Sephadex G‐50 column chromatography (CPMB UNIT ), ethanol precipitation of DNA ( appendix 3C), and labeling of DNA by random oligonucleotide–primed synthesis ( appendix 3E)
CAUTION: Hybridization solution and 104 cpm, 3′‐end‐labeled molecular size markers are hazardous; see appendix 2D guidelines on handling storage and disposal.NOTE: Experiments involving RNA require careful technique to prevent RNA degradation; see appendix 2A.

Support Protocol 1: Removal of Highly Repetitive Sequences from DNA Probes Using DNA‐Cellulose

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 100 µg/ml tested DNA‐cellulose (second support protocol and third support protocol support protocols)
  • TE buffer, pH 7.0
  • recipeCellulose prehybridization solution (see recipe)
  • 100 ng radiolabeled cDNA probe (basic protocol, step )
  • recipeCellulose hybridization solution (see recipe)
  • recipe2× SSPE, pH 7.0 (see recipe)
  • Cellulose regeneration solution: 99% (v/v) formamide/ 10 mM Tris⋅Cl (pH 7.5), freshly prepared
  • IEC HN‐SII centrifuge and 958 rotor (or equivalent)
  • 42° and 80°C water baths
CAUTION: Cellulose prehybridization solution, radiolabeled cDNA, cellulose hybridization solution, and formamide are hazardous; see appendix 2 for guidelines on handling, storage, and disposal.

Support Protocol 2: Preparation of DNA‐Cellulose

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • recipeAmmoniacal copper (II) hydroxide (ACH) solution, freshly prepared (see recipe)
  • m‐Aminobenzyloxymethyl‐cellulose (ABM‐cellulose; Sigma)
  • 30% (w/w) ammonium hydroxide
  • 95.8% sulfuric acid
  • 1.2 M HCl, freshly prepared and ice cold
  • 10 mg/ml sodium nitrate, freshly prepared
  • Urea
  • recipe0.5 M and 5 M sodium acetate buffer, pH 4.0, autoclaved (see recipe)
  • recipe0.1 M sodium acetate buffer (pH 4.0; see recipe)/80% (v/v) DMSO, room temperature and prewarmed to 50°C
  • 1 mg/0.9 ml high‐molecular‐weight genomic DNA ( appendix 3B)
  • Dimethylsulfoxide (DMSO)
  • recipe1× SSPE (see recipe), room temperature
  • 99% (v/v) formamide/0.1% (w/v) SDS, freshly prepared and prewarmed to 80°C
  • TE buffer, pH 7.0 ( appendix 2D)
  • 50‐ml centrifuge tube
  • IEC HN‐SII centrifuge and 958 rotor (or equivalent)
  • 50°, 70°, and 80°C water baths
  • Potassium iodide/starch test paper (Fisher)
  • 10‐ml tube, snap‐cap
  • Additional reagents and equipment for quantitation of DNA by absorption spectroscopy ( appendix 3D)
CAUTION: Formamide and 95.8% sulfuric acid are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 3: Testing DNA‐Cellulose

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 200 ng high‐molecular‐weight genomic DNA ( appendix 3B)
  • protocol 3100 µg DNA‐cellulose (second protocol 3support protocol)
  • Duplicate membrane filters containing phage or cosmid library of genomic DNA
  • recipeHybridization solution (see recipe)
  • 2× SSC/0.1% (w/v) SDS ( appendix 2)
  • 0.5× SSC/0.1% (w/v) SDS ( appendix 2)
  • 42° and 68°C water baths
  • Additional reagents and equipment for labeling DNA by nick translation ( appendix 3E)
CAUTION: Hybridization solution is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: High‐molecular‐weight genomic DNA, genomic library, and DNA coupled to cellulose should all be from the same species.
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Figures

Videos

Literature Cited

Literature Cited
   Brison, O., Ardeshir, F., and Stark, G.R. 1982. General method for cloning amplified DNA by differential screening with genomic probes. Mol. Cell. Biol. 2:578‐587.
   Dunne, P.W., Wang, S.‐W., Ashizawa, T., Perryman, M.B., and Epstein, H.F. 1992. cDNA surveying of specific tissue expression of human chromosome 19 sequences. Genomics 14:263‐269.
   Goldberg, M.L., Lifton, R.P., Stark, G.R., and Williams, J.G. 1979. Isolation of specific RNAs using DNA covalently linked to diazobenzyloxymethyl cellulose or paper. Methods Enzymol. 68:206‐220.
   Hochgeschwender, U. 1992. Toward a transcriptional map of the human genome. Trends Genet. 8:41‐44.
   Hochgeschwender, U. and Brennan, M.B. 1991. Identifying genes within the genome: New ways for finding the needle in a haystack. BioEssays 13:139‐144.
   Hochgeschwender, U., Sutcliffe, J.G., and Brennan, M.B. 1989. Construction and screening of a genomic library specific for mouse chromosome 16. Proc. Natl. Acad. Sci. U.S.A. 86:8482‐8486.
   Noyes, B.E. and Stark, G.R. 1975. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell 5:301‐310.
   den Van Ouweland, A.M.W., Kioschis, P., Verdijk, M., Tamanini, F., Toniolo, D., Poustka, A., and van Oost, B.A. 1992. Identification and characterization of a new gene in the human Xq28 region. Hum. Mol. Genet. 1:269‐273.
Key References
   Brison et al., 1982. See above.
  Describes use of DNA‐cellulose to remove highly repetitive sequences from genomic probes.
   Hochgeschwender et al., 1989. See above.
  Describes identification of genomic clones carrying sequences transcribed in different tissues by direct screening of an arrayed genomic library with cDNA as described in the basic protocol.
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