Gene2Oligo: oligonucleotide design for in vitro gene synthesis

Abstract

There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

Figure 1

An overview of the experimental process from the input sequence to the final in vitro synthesized gene.

Figure 2

Analysis of the synthetic CYB5 gene. About 10 µl of each sample are analyzed on a 1% agarose gel. Lanes: (L) 100 bp DNA ladder (NEB); (1) negative control, second PCR without template DNA; (2) LCR product before second PCR; (3) second PCR without primer after LCR; (4) second PCR after LCR; (5) assembly PCR product before second PCR; (6) second PCR without primer after assembly PCR; (7) second PCR after assembly PCR; and (8) positive control, second PCR using yeast genomic DNA as template. The arrow indicates the 388 bp CYB5 PCR product.