cDNA Amplification Using One‐Sided (Anchored) PCR

Robert L. Dorit1, Osamu Ohara2

1 Yale University, New Haven, Connecticut, 2 Shionogi Research Laboratories, Osaka, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.24
DOI:  10.1002/0471142735.im1024s08
Online Posting Date:  May, 2001
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Abstract

This unit presents a modification of PCR, called anchored PCR, that allows amplification of full‐length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.

     
 
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Table of Contents

  • Basic Protocol 1: Amplification of Regions Downstream (3′) of Known Sequence
  • Basic Protocol 2: Amplification of Regions Upstream (5′) of Known Sequence
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Amplification of Regions Downstream (3′) of Known Sequence

  Materials
  • Source of RNA (unit 10.11)
  • recipe5× Moloney murine leukemia virus (MoMuLV) reverse transcriptase buffer
  • 5 µg/µl bovine serum albumin (BSA)
  • 10 mM 4dNTP mix (10 mM each dNTP in TE buffer, pH 7.5, stored at −20°C)
  • 500 ng/µl actinomycin D (Boehringer‐Mannheim)
  • 200 U/µl MoMuLV reverse transcriptase (Boehringer‐Mannheim)
  • 15 pmol/µl (100 ng/µl) oligo(dT) 20 primer
  • 100 pmol/µl each of sequence‐specific primers 1 and 2 (see Fig. )
  • TE buffer, pH 7.5 ( appendix 2A)
  • 2.5 mM 4dNTP mix (2.5 mM each dNTP in TE buffer, pH 7.5, stored at −20°C)
  • recipe10× amplification buffer
  • 2.5 U/µl Taq DNA polymerase (unit 10.20)
  • Mineral oil
  • Additional reagents and equipment for preparation of poly(A)+ RNA (unit 10.11), PCR amplification (unit 10.20), agarose gel electrophoresis (unit 10.4), and Southern blotting and hybridization (unit 10.6)

Basic Protocol 2: Amplification of Regions Upstream (5′) of Known Sequence

  Materials
  • Source of RNA (unit 10.11)
  • 100 pmol/µl sequence‐specific primers 3 and 4 (Fig. )
  • 15 pmol/µl (100 ng/µl) oligo(dT) 20 primer
  • 1 M NaCl
  • 200 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 25 mM EDTA
  • 100% and 70% ethanol, ice cold
  • recipe5× Moloney murine leukemia virus (MoMuLV) reverse transcriptase buffer
  • 5 µg/µl bovine serum albumin (BSA)
  • 10 mM 4dNTP mix (10 mM each dNTP in TE buffer, pH 7.5, stored at −20°C)
  • 500 ng/µl actinomycin D (Boehringer‐Mannheim)
  • 200 U/µl MoMuLV reverse transcriptase (Boehringer‐Mannheim)
  • 3 M sodium acetate ( appendix 2A)
  • TE buffer, pH 7.5 ( appendix 2A)
  • recipe5× terminal deoxynucleotidyltransferase (TdT) buffer
  • 15 mM CoCl 2
  • 1 mM dATP (U.S. Biochemicals)
  • Terminal transferase (New England Biolabs or Boehringer‐Mannheim)
  • 40°C water bath
  • Additional reagents and equipment for preparation of poly(A)+ RNA (unit 10.11), phenol extraction (unit 10.1), PCR amplification (unit 10.20), and agarose gel electrophoresis (unit 10.4)
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Figures

Videos

Literature Cited

Literature Cited
   Dorit, R.L., Ohara, O., and Hwang, C. B.‐C. 1991. Direct DNA sequencing of PCR products. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds). pp. 15.2.1‐15.2.11. Greene Publishing Associates and John Wiley & Sons, New York.
   Frohman, M.A., Dush, M.K., and Martin, G.R. 1988. Rapid production of full length cDNAs from rare transcripts: Amplification using a single gene‐specific oligonucleotide primer. Proc. Nat. Acad. Sci. U.S.A. 85:8998‐9002.
   Holton, T.A. and Graham, M.W. 1991. A simple and direct method for direct cloning of PCR products using ddT‐tailed vectors. Nucl. Acids Res. 19:1156.
   Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. 1989. Polymerase chain reaction with single‐sided specificity: Analysis of a T‐cell receptor delta chain. Science 243:217‐220.
   Marchuck, D., Drumm, M., Saulino, A., and Collins, F.S. 1991. Construction of T‐vectors: A rapid and general system for direct cloning of unmodified PCR products. Nucl. Acids Res. 19:1154.
   Mueller, P.R. and Wold, B. 1989. In vivo footprinting of a muscle‐specific enhancer by ligation‐mediated PCR. Science 246:780‐786.
   Mueller, P.R., Garrity, P.A., and Wold, B. 1992. Ligation‐mediated PCR for genomic sequencing and footprinting. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds). pp. 15.5.1‐15.5.26. Greene Publishing Associates and John Wiley & Sons, New York.
   Natarajan, K., Burstyn, D., and Zauderer, M. 1992. Major histocompatibility complex determinants select T‐cell receptor α chain variable region dominance in a peptide‐specific response. Proc. Natl. Acad. Sci. U.S.A. 89:887‐8878.
   Ochman, H., Gerber, A.S., and Hartl, D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621‐623.
   Ohara, O., Dorit, R.L., and Gilbert, W. 1989. One‐sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad. Sci. U.S.A. 86:5673‐5677.
   Struhl, K. 1987. Subcloning of cDNA fragments. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds). pp. 3.16.1‐3.16.11. Greene Publishing Associates and John Wiley & Sons, New York.
   Tam, A.W., Smith, M.M., Fry, K.E., and Larrick, J.W. 1989. Construction of cDNA libraries from small numbers of cells using sequence independent primers. Nucl. Acids Res. 17:1269.
   Weis, J.H. 1987. Plating and transferring cosmid and plasmid libraries. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds). pp. 6.2.1‐6.2.3. Greene Publishing Associates and John Wiley & Sons, New York.
Key Reference
   Ohara, O., et al. 1989. See above.
  The protocols described here are used to isolate and characterize α‐tropomyosin transcripts in common frog and zebra fish.
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