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PCR Assembly of Synthetic Promoters

  • Hodan Mohamed
  • David GouldEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1651)

Abstract

In this chapter, we describe a two-step assembly PCR method to construct synthetic promoters. Essentially, this method takes advantage of specific annealing between complimentary DNA sequences to build random TFBS combinations within the assembled PCR products. A DNA polymerase is then employed to fill in the unpaired nucleotides in the generated sequences and also to amplify the assembled PCR products. We have used this method to generate synthetic promoters whereby the orientation of the TFBS can be controlled, the spacing between TFBS can be predetermined, and also the full diversity of the consensus TFBS can be covered through the use of degenerate oligonucleotides.

Key words

Assembly PCR Synthetic promoters Random assembly Multi-responsive promoters 

References

  1. 1.
    Dillon PJ, Rosen CA (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. BioTechniques 9(298):300Google Scholar
  2. 2.
    Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53CrossRefGoogle Scholar
  3. 3.
  4. 4.
    Mohamed H, Chernajovsky Y, Gould D (2016) Assembly PCR synthesis of optimally designed, compact, multi-responsive promoters suited to gene therapy application. Sci Rep 6:29388CrossRefGoogle Scholar
  5. 5.
    Wingender E, Chen X, Hehl R, Karas H, Liebich I, Matys V, Meinhardt T, Pruss M, Reuter I, Schacherer F (2000) TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res 28:316–319CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Public Health EnglandLondonUK
  2. 2.Department of Biochemical Pharmacology, William Harvey Research InstituteQueen Mary University of LondonLondonUK

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