Conversion of mRNA into Double‐Stranded cDNA

Lloyd B. Klickstein1, Rachael L. Neve2, Erica A. Golemis3, Jeno Gyuris4

1 Brigham and Women's Hospital, Boston, Massachusetts, 2 McLean Hospital, Belmont, Massachusetts, 3 Fox Chase Cancer Center, Philadelphia, Pennsylvania, 4 Mitotix, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.5
DOI:  10.1002/0471142727.mb0505s29
Online Posting Date:  May, 2001
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Abstract

Enzymatic conversion of mRNA into double‐stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide‐primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt‐ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The takes advantage of a linker‐primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt‐ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5‐methyl‐dCTP in the first‐strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI /XhoI ‐digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker‐primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.

     
 
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Table of Contents

  • Section III: Preparation of Insert DNA From Messenger RNA
  • Basic Protocol 1: Conversion of mRNA into Blunt‐Ended Double‐Stranded cDNA
  • Alternate Protocol 1: Conversion of mRNA into Double‐Stranded cDNA for Directional Cloning
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Conversion of mRNA into Blunt‐Ended Double‐Stranded cDNA

  Materials
  • 5 mM 4dNTP mix (unit 3.4)
  • recipe5× reverse transcriptase (RT)buffer (see recipe)
  • 200 mM dithiothreitol (DTT)
  • 0.5 mg/ml oligo(dT)12–18 (Pharmacia Biotech; store at −80°C) or 15‐ to 40‐mer antisense primer or random‐hexamer primers
  • RNasin ribonuclease inhibitor (Promega; store at −20°C)
  • AMV (avian myeloblastosis virus) reverse transcriptase (Life Sciences; unit 3.7)
  • 10 µCi/µl [α‐32P]dCTP (10,000 Ci/mmol)
  • 0.5 M EDTA, pH 8.0
  • Buffered phenol (unit 2.1)
  • TE buffer, pH 7.5 ( appendix 22)
  • Diethyl ether or 24:1 chloroform/isoamyl alcohol
  • 7.5 M ammonium acetate
  • 95% and 70% ethanol, ice‐cold
  • 10% trichloroacetic acid (TCA), ice‐cold
  • recipe5× second‐strand buffer I (see recipe)
  • 5 mM β‐NAD+ (Sigma; store at −80°C)
  • RNase H (Pharmacia Biotech; unit 3.13)
  • E. coli DNA ligase (New England Biolabs; unit 3.14)
  • E. coli DNA polymerase I (New England Biolabs; unit 3.5)
  • recipe5× TA buffer (see recipe)
  • recipe2 µg/ml RNase A, DNase‐free (see recipe and unit 3.13)
  • T4 DNA polymerase (Boehringer Mannhein; unit 3.5)
  • 10 mg/ml tRNA (store at −20°C)
  • 42° and 65°C water baths
  • Nitrocellulose membrane filter
  • 14°C incubator
  • Additional reagents and equipment for preparation of poly(A)+ RNA (unit 4.5) and purification and concentration of DNA (unit 2.1)

Alternate Protocol 1: Conversion of mRNA into Double‐Stranded cDNA for Directional Cloning

  • recipe5× SuperScript buffer (RNase‐free; see recipe)
  • 0.1 M DTT (RNase‐free)
  • 3dNTP/methyl‐dCTP mix: 10 mM each dATP, dGTP, dTTP, and 5‐methyl‐dCTP (Pharmacia Biotech)
  • 0.25 µg/µl oligonucleotide primer (unit 2.11) incorporating (from 5′ to 3′): (dGdA) 10, XhoI restriction site, and (dT) 18
  • 200 U/µl SuperScript or SuperScript II (GIBCO/BRL)
  • recipe5× second‐strand buffer II (see recipe)
  • 10 mM and 2 mM 4dNTP mix (Pharmacia Biotech)
  • 10 µCi/µl [α‐32P]dATP (3000 Ci/mmol)
  • 0.8 U/µl RNase H (Pharmacia Biotech)
  • 10 U/µl E. coli DNA polymerase I (New England Biolabs)
  • 1:1 (w/v) phenol/chloroform
  • 100% ethanol ice‐cold
  • recipe10× Klenow buffer (see recipe)
  • 5 U/µl Klenow fragment of E. coli DNA polymerase I (New England Biolabs)
  • recipe10× mung bean nuclease buffer (see recipe)
  • 10 U/µl mung bean nuclease (New England Biolabs)
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 22)
  • 16°C incubator
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Gubler, U. and Hoffman, B.J. 1983. A simple and very effective method for generating cDNA libraries. Gene 25:263‐269.
   Kotewicz, M.L., Sampson, C.M., D'Alessio, J.M., and Gerard, G.F. 1988. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucl. Acids Res. 16:265‐277.
   Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
   Neve, R.L., Hanis, P., Kosik, K.S., Kurnit, D.M., and Donlon, T.A. 1986. Identification of the gene for the human microtubule‐associated protein tau and chromosomal localization of the genes for tau and microtubule‐associated protein 2. Brain Res. 387(3):271‐80.
   Okayama, H. and Berg, P. 1982. High‐efficiency cloning of full‐length cDNA. Mol. Cell. Biol. 2:161‐170.
   Tamkun, J.W., DeSimone, D.W., Fonda, D., Patel, R.S., Buck, D., Horwitz, A.F., and Hynes, R.O. 1986. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell 46:271‐282.
Key References
   Gubler and Hoffman, 1983. See above
  First demonstrated use of linker primers and methylated nucleotides in cDNA synthesis.
   Huse, W.D. and Hansen, C. 1988. cDNA cloning redefined. Strategies (Stratagene) 1:1‐3.
  These authors first developed the RNase H/E. coli DNA polymerase I alternative to S1 nuclease and Klenow fragment for second‐strand synthesis.
   Okayama and Berg, 1982. See above.
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