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 Molecular Biology
Unit Number:  Unit 15.6
DOI:  10.1002/0471142727.mb1506s17
Online Posting Date:  May, 2001
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Abstract

The polymerase chain reaction (PCR) offers an opportunity to directly select, amplify, and isolate a message of interest. This unit describes anchored PCR, a modification of the standard reaction that allows amplification of full‐length mRNA when only a small amount of sequence information is available. Anchored PCR can be employed when only a small region of sequence lying within the mRNA is known in advance, unlike the amplification of RNA or cDNA by conventional PCR which requires prior knowledge of the sequences flanking the region of interest to design the PCR primers.

     
 
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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 (units 4.1 4.5)
  • recipe5× Moloney murine leukemia virus (MoMLV) 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
  • 200 U/µl MoMLV reverse transcriptase (Table 97.80.4711 & unit 3.7)
  • 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 ( appendix 22)
  • 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 (units 15.1 & 3.5)
  • Mineral oil
  • Additional reagents and equipment for preparation of poly(A)+ RNA (unit 4.1), PCR amplification (unit 15.1), agarose gel electrophoresis (unit 2.5), and Southern blotting and hybridization (units 2.9 & 6.4)

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

  Materials
  • Source of RNA (units 4.1 4.5)
  • 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 22)
  • 25 mM EDTA
  • 100% and 70% ethanol, ice‐cold
  • recipe5× Moloney murine leukemia virus (MoMLV) 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
  • 200 U/µl MoMLV reverse transcriptase (Table 97.80.4711 & unit 3.7)
  • 3 M sodium acetate ( appendix 22)
  • TE buffer, pH 7.5 ( appendix 22)
  • recipe5× terminal deoxynucleotidyltransferase (TdT) buffer
  • 15 mM CoCl 2
  • 1 mM dATP (unit 3.4)
  • Terminal transferase (Table 97.80.4711 & unit 3.6)
  • 40°C water bath
  • Additional reagents and equipment for preparation of poly(A)+ RNA (unit 4.5), phenol extraction (unit 2.1), and PCR amplification (unit 15.1)
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Figures

Videos

Literature Cited

Literature Cited
   Frohman, M.A., Dush, M.K., 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.
   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.
   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.
Key Reference
   Ohara 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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