Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides

David D. Moore1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.2B
DOI:  10.1002/0471142727.mb0802bs26
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This protocol uses pairs of oligonucleotides annealed at a short duplex segment at their 3' ends as both templates and primers (mutually primed synthesis) to generate desired sequences up to 400 bp in a single step. The procedure is divided into three sections: design of the oligonucleotides, extension by mutually primed synthesis, and cloning of the extension products. The strategy for design of oligonucleotides is discussed in the commentary.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • 10× T7 DNA polymerase (Sequenase) buffer (unit 3.4)
  • 4dNTP mix (2.5 mM each dNTP; unit 3.4)
  • Modified T7 DNA polymerase (Sequenase, U.S. Biochemical; unit 3.5)
  • 4 M ammonium acetate
  • 100% and 95% ethanol
  • TE buffer
  • Additional reagents and equipment for restriction endonuclease cleavage (unit 3.1), phenol extraction and ethanol precipitation (unit 2.1), sieving agarose gel electrophoresis (unit 2.8), subcloning (unit 3.1), and DNA sequencing (unit 7.4)
  • The following steps replace steps to of the protocol 1 in unit 8.2.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aota, S.I., Gojobi, T., Ishibashi, F., Maruyama, T., and Ikemura, T. 1988. Codon usage compiled from the GenBank sequence data. Nucl. Acids Res. 16:r315‐r403.
   Devereaux, J., Haberli, P. and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12:387‐395.
   Kozak, M. 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196:947‐950.
   Uhlmann, E., 1988. An alternate approach in gene synthesis: Use of long selfpriming oligodeoxynucleotides for construction of double‐stranded DNA. Gene 71:29‐40.
   Wosnick, M.A., Barnett, R.W., Vicentini, A.M., Erfle, H., Elliott, R., Summner‐Smith, M., Mantei, N., and Davies, R.W. 1988. Rapid construction of large synthetic genes: Total chemical synthesis of two different versions of the bovine prochymosin gene. Gene 60:115‐127.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library