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Construction of Synthetic Genes by Polymerase Chain Reaction

  • Patrick J. Dillon
  • Craig A. Rosen

Abstract

Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a simple two-step PCR method for the rapid construction of synthetic genes (3). This method is based on early observations by Mullis et al. (4) in which multiple overlapping oligonucleotides could be used to generate synthetic DNA through several sequential rounds of Klenow based PCR amplification. The method described in this chapter utilizes the thermostable Taq polymerase and allows for the generation of synthetic genes in as little as 1 d. This method has proven useful in studies in which synthetic genes were constructed for the HIV-2 Rev protein (3,5) and the Wilms’ tumor locus zinc finger protein (6). Furthermore, this method has been successfully employed in extensive mutagenesis of the HIV-1 rev response element (7).

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Reaction Polymerase Chain Reaction Buffer Nucleic Acid Sequence Synthetic Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

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    Dillon, P. J., Nelbock, P., Perkins, A., and Rosen, C. A. (1990) Function of the human immunodeficiency virus types I and 2 Rev proteins is dependent upon their ability to interact with a structural region present in the env gene mRNA. J. Virol. 64, 4428–4437.PubMedGoogle Scholar
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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Patrick J. Dillon
    • 1
  • Craig A. Rosen
    • 2
  1. 1.Research and DevelopmentInvitrogen, Carlsbad
  2. 2.Research and DevelopmentHuman Genome SciencesRockville

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