Oli2go: an automated multiplex oligonucleotide design tool

Abstract

The success of widely used oligonucleotide-based experiments, ranging from PCR to microarray, strongly depends on an accurate design. The design process involves a number of steps, which use specific parameters to produce high quality oligonucleotides. Oli2go is an efficient, user friendly, fully automated multiplex oligonucleotide design tool, which performs primer and different hybridization probe designs as well as specificity and cross dimer checks in a single run. The main improvement to existing oligonucleotide design web-tools is that oli2go combines multiple steps in an all-in-one solution, where other web applications only accomplish parts of the whole design workflow. Especially, the oli2go specificity check is not only performed against a single species (e.g. mouse), but against bacteria, viruses, fungi, invertebrates, plants, protozoa, archaea and sequences from whole genome shotgun sequence projects and environmental samples, at once. This allows the design of highly specific oligonucleotides in multiplex applications, which is further assured by performing dimer checks not only on the primers themselves, but in an all-against-all fashion. The software is freely accessible to all users at http://oli2go.ait.ac.at/.

Phytohormonal cross-talk modulate Bipolaris sorokiniana (Scc.)interaction with Zea mays

“…In order to normalise the expression level of target genes, expression of housekeeping genes ( DPP9 ) was compared (Amaral et al, 2017). The primers for genes (Table S1) were designed using online Oli2go (http://oli2go.ait.ac.at/) on desired temperature and primer length (Hendling et al, 2018).…”

Optimization of the experimental parameters of the ligase cycling reaction

“…One important parameter for the LCR assembly is the melting temperature ( T m ) of the BOs at around for each half to facilitate optimal hybridization of template and oligonucleotide for given cycling parameters. Closely related is the free energy , which is assumed as the more important quantity for oligonucleotide-based biological experiments ( 12 , 13 ). In the LCR, its impact is counteracted by using dimethyl sulfoxide (DMSO) and betaine to increase and thus to reduce secondary structures ( 9 , 10 ).…”

“…Bode et al ( 15 ) offers similar functionality. Another web-application includes the design of primers and performs T m and cross-checks for the oligonucleotide sequences against themselves, their DNA probes and whole genomes ( 13 ) but is not applied for the LCR. Robinson et al ( 16 ) use a BO-design-tool with an adjustable target melting temperature but without optimizing the crosstalk.…”

Optimization of the experimental parameters of the ligase cycling reaction

“…One important parameter for the LCR-assembly is the melting temperature (T m ) of the BOs at around 70 • C for each half to facilitate optimal hybridization of template and oligonucleotide for given cycling parameters. Closely related is the free energy ∆G, which is assumed as the more important quantity for oligonucleotide-based biological experiments (12,13). In the LCR, its impact is counteracted by using dimethyl sulfoxide (DMSO) and betaine to increase ∆G and thus to reduce secondary structures (8,9).…”

“…Bode et al (15) offers similar functionality. Another web-application includes the design of primers and performs T m and ∆G cross-checks for the oligonucleotide sequences against themselves, their DNA probes and whole genomes (13) but is not applied for the LCR. Robinson et al (16) use a BO-design-tool with an adjustable target melting temperature but without optimizing the crosstalk.…”