Gene2Oligo: oligonucleotide design for in vitro gene synthesis.
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.
Rouillard
Jean-Marie
JM
Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA. jmrouill@umich.edu
Lee
Woonghee
W
Truan
Gilles
G
Gao
Xiaolian
X
Zhou
Xiaochuan
X
Gulari
Erdogan
E
D000465:Algorithms; D015786:Cytochromes b5 / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D005800:Genes, Fungal; D020407:Internet; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000096:biosynthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software; D013696:Temperature
D009841:Oligonucleotides; D015786:Cytochromes b5; D011088:DNA Ligases