CH391L/S12/GeneandGenomeSynthesis

Gene and Genome Synthesis

Introduction

Purpose, needs, overview. Most sequences today are assembled in part or in whole from naturally occurring sequences. This limits applications and interchangeability of components in synthetic gene constructs. It's all about oligos: scale, cost, fidelity, assembly

History of Gene Synthesis

tRNA gene, first one took 5 years Oligo synthesis (basic chemistry)

Summary of Methods

Ligation-Based Methods

In ligation-mediated assembly, the synthetic gene or DNA fragment of interest is broken up into individual overlapping DNA oligonucleotides that cover both strands of the eventual DNA duplex. In contrast to PCR-based methods of gene synthesis, there are no gaps introduced in either strand during design, but rather the oligonucleotides completely reconstitute the eventual DNA target.

The oligonucleotides are chemically synthesized and phosphorylated at their 5' ends using purified kinase in an in vitro reaction. After complementary oligo fragments are annealed using using thermal cycling, purified DNA ligase is added to the reaction in order to splice together the 3' OH and 5' phosphate gaps on each backbone.

PCR-based Methods

PCA

Solid-Phase Synthesis

Blue Heron

Multiplex/Microchip Synthesis

Oligos are commodity, still expensive on large scale Microarray based oligo synth - Inkjets, photolithography, electrochemical Chips allow large numbers of oligos, small scale. On-chip amplification with universal PCR primers in frags can amplify pools to levels useful in gene synthesis methods. Oligo error rates reduced in multiplex using hybridization screening arrays to eliminate mismatched oligos. Nonselected oligo synthesis methods have error rate of 1 in ~160 bp, selected multiplex oligo methods have 1 in ~1,500 error rate. Further work could extend this to 1 in 10^6 using mismatch proteins. Multiplex uses: ITT protein arrays, synthesis of operons (Tian 2004 ribosomal operon from E. coli, 21 genes and codon optimization)