High-Quality Gene Synthesis With Microchip-Synthesized Oligonucleotides
To meet the growing demand for synthetic genes, better quality, scalable and inexpensive gene assembly technologies should be developed. Methods for large-scale, high-quality gene synthesis at a reasonable price are essential for advances in both synthetic biology and biotechnology.
One limitation for gene synthesis is the cost of making the foundations (oligonucleotides) that are assembled together to create genes. Current oligonucleotide synthesis methods via standard solid support synthesis cost ~ $0.20/bp. Synthesizing oligonucleotides on DNA microchips (microarrays) has got the potential to greatly increase throughput - and therefore reduce cost - compared with current column synthesis methods MGB probe.
However, microchip-based synthesis results in complex mixtures of unpurified oligonucleotides, leading to difficulties in assembling gene fragments and potential cross-hybridization between assembled fragments. The notion of employing a "selection" method incorporated in the gene synthesis protocol to eliminate the incorporation of oligonucleotides containing undesirable synthesis errors was initially introduced 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 reduced total of error rates in synthetic genes prepared from crude oligo mixtures. The initial describes the utilization 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 initial 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 all the way 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 utilizing the new protocol matched the caliber of the genes assembled from >95% pure column-synthesized oligonucleotides by the typical protocol and genes assembled from microchip-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones.
One limitation for gene synthesis is the cost of making the foundations (oligonucleotides) that are assembled together to create genes. Current oligonucleotide synthesis methods via standard solid support synthesis cost ~ $0.20/bp. Synthesizing oligonucleotides on DNA microchips (microarrays) has got the potential to greatly increase throughput - and therefore reduce cost - compared with current column synthesis methods MGB probe.
However, microchip-based synthesis results in complex mixtures of unpurified oligonucleotides, leading to difficulties in assembling gene fragments and potential cross-hybridization between assembled fragments. The notion of employing a "selection" method incorporated in the gene synthesis protocol to eliminate the incorporation of oligonucleotides containing undesirable synthesis errors was initially introduced 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 reduced total of error rates in synthetic genes prepared from crude oligo mixtures. The initial describes the utilization 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 initial 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 all the way 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 utilizing the new protocol matched the caliber of the genes assembled from >95% pure column-synthesized oligonucleotides by the typical protocol and genes assembled from microchip-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones.
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