Gene Synthesis

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Tian et al.: Accurate multiplex gene synthesis from programmable DNA microchips. Nature 2004;432:1050-4.

The above paper describes a multi-stage process for synthesizing, error-proofing and assembling oligonucleotide fragments, using photoprogrammable microfluidic chips and PCR.

The process is summarized below.

Please edit, or add comments to, this page if you feel moved to do so.

  • Background:
    • DNA synthesis is currently too expensive for whole-genome synthesis
    • Currently costs $.11/base (short oligos)
    • High error rate (deletions every 100bp, substitutions/insertions every 400bp)
    • With proofreading/error-correction/labor, goes up to $2/base
  • Links:

Comments:

  • not hard to see that cheap DNA synthesis will change the world
  • when microarrays arrived in the 90's, academic labs were quick to replicate the technology

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Tian et al.: Accurate multiplex gene synthesis from programmable DNA microchips. Nature 2004;432:1050-4.

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Outline of process

  1. Computer-aided sequence optimization of codon usage, restriction enzyme sites and mRNA secondary structure
    • program used is called CAD-PAM
  1. "Construction oligo" synthesis using Xeotron microfluidics and photolithography
    • Seems to be basically the same as making a microarray - Ian Holmes
  1. Proofreading of construction oligos using two pools of bead-immobilized complementary "selection oligos"
    • Selection oligos designed to have uniform melting temperatures from the double-stranded state
  1. The polymerase assembly multiplexing reaction (PAM). It is proposed that this single step can replace the "traditional" three-step ligation-assembly-PCR. Requires:
    • Construction oligos
    • Gene-flanking primer pairs (high concentration)
    • Thermostable polymerase
    • dNTP's
    • Thermal cycler

Here are the three papers on oligo assembly that are cited:

  1. Cello et al.: Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 2002;297:1016-8.
  2. Smith et al.: Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 2003;100:15440-5.
  3. Stemmer et al.: Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 1995;164:49-53.