Molecular Cloning of PCR Products

Michael Finney1, Paul E. Nisson2, Ayoub Rashtchian2

1 MJ Research, Watertown, Massachusetts, 2 Life Technologies, Inc., Gaithersburg, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.4
DOI:  10.1002/0471142727.mb1504s56
Online Posting Date:  November, 2001
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Abstract

It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high‐quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.

     
 
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Table of Contents

  • Basic Protocol 1: Generation of T‐A Overhangs
  • Alternate Protocol 1: Generation of Half‐Sites
  • Alternate Protocol 2: Cloning PCR Products with Uracil DNA Glycosylase
  • Support Protocol 1: Designing Primer Sets for Amplification and Construction of UDG Cloning Vectors
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of T‐A Overhangs

  Materials
  • 5 µg vector DNA—e.g., pUC19 (unit 1.5) or M13mp18 (unit 1.14)
  • TE buffer, pH 8.0 ( appendix 22)
  • 10× PCR buffer (with optimized Mg2+ concentration, see unit 15.1)
  • 5 mM dTTP (unit 3.4)
  • 5 U/µl Taq DNA polymerase
  • Target DNA
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), purification of DNA (unit 2.1), PCR (unit 15.1), and subcloning DNA fragments (unit 3.16)

Alternate Protocol 1: Generation of Half‐Sites

  • T4 DNA ligase (unit 3.14) and 10× buffer (unit 3.4)
  • 1 mM ATP (unit 3.4)
  • 2 mM 4dNTP mix (unit 15.1)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • T4 polynucleotide kinase (unit 3.10)
  • Additional reagents and equipment for oligonucleotide synthesis (unit 2.11) and quantitation of DNA ( appendix 3D)

Alternate Protocol 2: Cloning PCR Products with Uracil DNA Glycosylase

  • PCR product amplified using dU‐containing primers (see protocol 4)
  • 25 ng/µl linearized UDG cloning vector (see protocol 4)
  • 5× PCR buffer (with optimized Mg2+ concentration; see unit 15.1)
  • 1 U/µl uracil DNA glycosylase (UDG)
  • Additional reagents and equipment for introduction of DNA into cells (unit 1.8), screening transformants (unit 1.4), mapping by restriction endonuclease digestion (unit 3.2 & )

Support Protocol 1: Designing Primer Sets for Amplification and Construction of UDG Cloning Vectors

  Materials
  • Vector DNA linearized at appropriate cloning or insertion site
  • DNA sequence to be amplified
  • Additional reagents and equipment for synthesis of oligonucleotides (unit 2.11), PCR (unit 15.1), and agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Aslandis, C. and de Jong, P.J. 1990. Ligation‐independent cloning of PCR products (LIC‐PCR). Nucl. Acids Res. 18:6069‐6074.
   Booth, P.M., Buchman, G.W., and Rashtchian, A. 1994. Assembly and coding sequences for neurotrophic factors directly from genomic DNA using polymerase chain reaction and uracil DNA glycosylase. Gene 146:303‐308.
   Buchman, G.W., Schuster, D.M., and Rashtchian, A. 1992. Rapid and efficient cloning of PCR products using the CloneAmp system. Focus 14:41‐45.
   Buchman, G.W., Booth, P., and Rashtchian, A. 1993. The CloneAmp pUC18 system: Cloning of the brain‐derived neurotrophic factor gene. Focus 15:36‐41.
   Clark, J.M. 1988. Novel nontemplated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nucl. Acids Res. 16:9677‐9686.
   Kaufman, D.L. and Evans, G.A. 1990. Restriction endonuclease cleavage at the termini of PCR products. BioTechniques 9:304‐306.
   Kovalic, D., Kwak, J.‐H., and Weisblum, B. 1991. General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucl. Acids Res. 19:4650.
   Marchuk, 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.
   Nisson, P.E., Rashtchian, A., and Watkins, P.C. 1991. Rapid and efficient cloning of Alu‐PCR products using uracil DNA glycosylase. PCR Methods Appl. 1:120‐123.
   Owen, J.L., Hay, C., Schuster, D.M., and Rashtchian, A. 1994. A highly efficient method of site‐directed mutagenesis using PCR and UDG cloning. Focus 16:39‐44.
   Rashtchian, A. 1995. Novel methods for cloning and engineering genes using the polymerase chain reaction. Curr. Opin. Biotechnol. 6:30‐36.
   Rashtchian, A., Buchman, G., Schuster, D., and Berninger, M. 1992a. Uracil DNA glycosylase–mediated cloning of PCR‐ampified DNA: Application to genomic and cDNA cloning. Anal. Biochem. 200:91‐97.
   Rashtchian, A., Thornton, C.G., and Heidecker, G. 1992b. A novel method for site‐directed mutagenesis using PCR and uracil DNA glycosylase. PCR Meth. Appl. 2:124‐130.
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