PCR‐Based Subtractive cDNA Cloning

Mukesh Patel1, Hazel Sive1

1 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.2
DOI:  10.1002/0471142727.mb25b02s55
Online Posting Date:  August, 2001
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Subtractive cloning is a powerful technique that allows isolation and cloning of mRNAs differentially expressed in two cell populations. In the generalized subtraction scheme the cell types to be compared are the [+] or tracer cells and the [‐] or driver cells, where mRNAs expressed in the tracer and not the driver are isolated. Briefly, tracer nucleic acid (cDNA or mRNA) from one cell population is allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences common to both cell populations. Hybrids between the tracer and driver, and all driver sequences, are removed in the subtraction step. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. The method described in this unit uses double‐stranded cDNA (ds cDNA) as both tracer and driver. Reciprocal subtractions are performed between two cell populations, A and B: that is, genes preferentially expressed in A more than in B are isolated, as are genes expressed preferentially in B more than in A. The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction. The progress of subtraction is monitored by slot blot hybridization. Differentially expressed cDNA sequences are used to construct a subtracted cDNA library.

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Table of Contents

  • Basic Protocol 1: Construction of Subtracted cDNA Libraries
  • Support Protocol 1: Slot Blot Hybridization to Monitor Subtraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Construction of Subtracted cDNA Libraries

  • Double‐stranded cDNA (ds cDNA) for cell types A and B (unit 5.5)
  • AluI and recipe10× AluI buffer (see recipe)
  • RsaI
  • 10, 15, and 75 mM ATP
  • 10 U/µl T4 polynucleotide kinase and recipe10× T4 polynucleotide kinase buffer (see recipe)
  • Oligonucleotide primers
  •  3 µg/µl a1: 5′‐TAG TCC GAA TTC AAG CAA GAG CAC A‐3′
  •  2.5 µg/µl a2: 5′‐CTC TTG CTT GAA TTC GGA CTA‐3′
  •  3 µg/µl b1: 5′‐ATG CTG GAT ATC TTG GTA CTC TTC A‐3′
  •  2.5 µg/µl b2: 5′‐GAG TAC CAA GAT ATC CAG CAT‐3′
  • 10 U/µl T4 DNA ligase and recipe10× T4 DNA ligase buffer (see recipe)
  • 40% (w/v) polyethylene glycol 8000 (PEG 8000)
  • 25:24 (v/v) phenol/chloroform (made with buffered phenol; unit 2.1)
  • Chloroform
  • 5 U/µl Taq DNA polymerase and recipe10× Taq DNA polymerase buffer (see recipe)
  • 25 mM MgCl 2
  • 10 mM 4dNTP mix (unit 3.4)
  • Mineral oil, PCR‐grade, sterile
  • 800 Ci/mmol [α32P]dCTP (10 Ci/µl)
  • recipeDriver dNTP mix (see recipe)
  • Ethanol
  • 1 and 5 M NaCl
  • recipeHEPES buffer (see recipe)
  • recipe2× hybridization buffer for subtractions (see recipe)
  • recipeStreptavidin solution (see recipe)
  • EcoRI and recipe10×EcoRI buffer (see recipe) or EcoRV and recipe10×EcoRV buffer (see recipe)
  • pBluescript vector cut with EcoRI
  • pBluescript vector cut with EcoRV
  • Tranformation‐competent bacterial strain (unit 1.8)
  • Radiolabeled subtraction probes (see protocol 2)
  • 0.5‐ml PCR tubes
  • Sephacryl S‐300 spin columns (Pharmacia Biotech)
  • Beckman Accuspin FR centrifuge with swinging‐bucket rotor or equivalent
  • Thermal cycler
  • Anion‐exchange PCR spin columns (Qiagen)
  • 1.5‐ml microcentrifuge tubes, silanized ( appendix 3B)
  • Hand‐held Geiger counter
  • Heating block
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), chromatography to remove oligonucleotide fragments (unit 2.6), phenol/chloroform extraction and ethanol precipitation (unit 2.1), anion‐exchange (Qiagen) column purification of oligonucleotides (unit 2.1), spectrophotometric quantitation of nucleic acids ( appendix 3D), hybridization of slot blots (unit 2.9 & ; also see protocol 2), bacterial transformation (unit 1.8), plating libraries (unit 6.1), preparing replica filters (unit 6.2), hybridizing replica filters (unit 6.3), preparing minipreps of plasmid DNA (unit 1.6), and sequencing plasmid DNA (units 7.4 & 7.4)

Support Protocol 1: Slot Blot Hybridization to Monitor Subtraction

  • cDNA from each subtraction (see protocol 1, step )
  • 3 M NaOH
  • 2 M ammonium acetate, pH 7.0
  • recipeProbe dNTP mix (see recipe)
  • Sephadex G50/80 spin column (Pharmacia Biotech) in sterile 1‐ml syringe
  • Additional reagents and equipment for slot blotting (unit 2.9) and hybridization (unit 2.10)
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Literature Cited

Literature Cited
   Buckbinder, L. and Brown, D.D. 1992. Thyroid hormone–induced gene expression changes in the developing frog limb. J. Biol. Chem. 267:25786‐25791.
   Davidson, E.H. 1986. Complexity of maternal RNA. In. Gene Activity in Early Development, 3rd ed., pp. 50‐55. Academic Press, San Diego.
   Duguid, J.R., Rohwer, R.G., and Seed, B. 1988. Isolation of cDNAs of scrapie‐modulated RNAs by subtractive hybridization of a cDNA library. Proc. Natl. Acad. Sci. U.S.A. 85:5738‐5742.
   Duguid, J.R. and Dinauer, M.C. 1989. Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucl. Acids Res. 18:2789‐2792.
   Hara, E., Yamaguchi, T., Tahara, H., Tsuyama, N., Tsurui, H., Ide, T., and Oda, K. 1993. DNA‐DNA subtractive cDNA cloning using oligo dT‐Latex and PCR: Identification of cellular genes which are overexpressed in senescent human diploid fibroblasts. Anal. Biochem. 214:58‐64.
   Patanjali, S.R., Parimoo, S., and Weissman, S.M. 1991. Construction of a uniform abundance (normalized) cDNA library. Proc. Natl. Acad. Sci. U.S.A. 88:1943‐1947.
   Rosenberg, M., Przylbylska, M., and Straus, D. 1994. RFLP subtraction: A method for making libraries of polymorphic markers. Proc. Natl. Acad. Sci. U.S.A. 91:6113‐6117.
   Rubenstein, J.L.R., Brice, A.E.J., Ciaranello, R.D., Denney, D., Porteus, M.H., and Usdin, T.B. 1990. Subtractive hybridization system using single‐stranded phagemids with directional inserts. Nucl. Acids Res. 18:4833‐4842.
   Sive, H.L. and St. John, T. 1988. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucl. Acids Res. 16:10937.
   Sive, H.L., Hattori, K., and Weintraub, H. 1989. Progressive determination during formation of anteroposterior axis in Xenopus laevis. Cell. 58:171‐180.
   Soares, M.B., Bonaldo, M.F., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A. 1994. Construction and characterization of a normalized cDNA library. Proc. Natl. Acad. Sci. U.S.A. 91:9228‐9232.
   Straus, D. and Ausubel, F.M. 1990. Genome subtraction for cloning DNA corresponding to deletion mutants. Proc. Natl. Acad. Sci. U.S.A. 87:1889‐1893.
   Uhlen, M. 1989. Magnetic separation of DNA. Nature. 340:733‐734.
   Wang, Z. and Brown, D.D. 1991. A gene expression screen. Proc. Natl. Acad. Sci. U.S.A. 88:11505‐11509.
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