High Fidelity
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High Fidelity
High-Quality Gene Synthesis with Microchip-Synthesized Oligonucleotides
To meet the growing demand for synthetic genes, more robust, scalable and inexpensive gene assembly technologies must be developed. Methods for large-scale, high-quality gene synthesis at an affordable price are needed for advances in both synthetic biology and biotechnology.
One limitation for gene synthesis is the cost of making the building blocks (oligonucleotides) that are assembled together to make genes. Current oligonucleotide synthesis methods via standard solid support synthesis cost ~ $0.20/bp. Synthesizing oligonucleotides on DNA microchips (microarrays) has the potential to greatly increase throughput — and therefore reduce cost — compared with current column synthesis methods.
However, microchip-based synthesis results in complex mixtures of unpurified oligonucleotides, which leads to difficulties in assembling gene fragments and potential cross-hybridization between assembled fragments. The idea of using a "selection" method incorporated in the gene synthesis protocol to eliminate the incorporation of oligonucleotides containing undesirable synthesis errors was first introduced back in 2004. Researchers then used microchip-synthesized oligonucleotides to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit1.
Two recent studies describe new approaches to reduction of error rates in synthetic genes prepared from crude oligo mixtures. The first describes the use of hybridization-based selection embedded in the assembly process2 and another introduces a method, called megacloning that utilizes next-generation sequencing (NGS) technology as a preparative tool3.
In the first study, researchers have eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 crude oligonucleotides eluted directly from microchips. The oligos were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microchip-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol and genes assembled from microchip-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones.
In the second study, researchers describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality synthetic DNA by using next-generation sequencing (NGS) technology as a preparative tool. Microchip-synthesized oligonucleotides are processed through an NGS run procedure to generate sequence-verified DNA clones. A robotic system is used for imaging and picking beads containing the clones directly off of a high-throughput pyrosequencing platform and the clones are used for subsequent gene assembly, avoiding the need for any other selection steps. The method reduced error rates by a factor of 500 compared to the starting crude oligonucleotide pool generated by microchip and the DNA obtained was used to assemble fully functional synthetic genes.
Approaches such as these reduce synthesis costs down to <$0.01/bp and will ultimately result in more widespread and economical construction of synthetic genomes and large gene libraries.
1. Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., and Church, G. (2004) Accurate multiplex gene synthesis from programmable DNA chips. Nature 432, 1050-1054. [abstract]
2. Borovkov AY, Loskutov AV, Robida MD, Day KM, Cano JA, Le Olson T, Patel H, Brown K, Hunter PD, Sykes KF. (2010) High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides. Nucleic Acids Res 38(19), e180. [article]
3. Matzas M, Stähler PF, Kefer N, Siebelt N, Boisguérin V, Leonard JT, Keller A, Stähler CF, Häberle P, Gharizadeh B, Babrzadeh F, Church GM. (2010) High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. Nat Biotechnol 28(12), 1291-94. [abstract]
About the Author
Chris Hebel
LC Sciences
2575 West Bellfort St #270
Houston, TX 77054
Tel. 713-664-7087
Fax. 713-664-8181
chebel@lcsciences.com
Is the a difference between the movie and book of high fidelity?
I just need to know if there are any like huge differences between Nick Hornby's book and John Cusack's movie high fidelity
Do you mean apart from one being set in england and the other in america?
High Fidelity trailer