A unique tool enabling low cost, multiplex custom oligonucleotides for various applications serving for construction of biomolecular libraries containing up to millions of different biomolecules of defined sequences. These products significantly reduce the cost and increase the speed of highly…

FISH utilizes fluorescent probes to bind portions of DNA that have a high degree of sequence complementarity. This allows researchers to detect and localize specific DNA sequences on chromosomes or RNA targets in various cell and tissue types to determine the spatial-temporal patterns of gene expression within. In medicine, FISH can be used to diagnose or evaluate the progression of a disease, such as cancer, to identify a particular species or to perform various types of karyotyping.

Developing a FISH assay requires the use of oligonucleotide probe sets, like oligopaint probes, which are fluorescently labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. LC Sciences’ OligoMix offers a unique solution for researchers looking to generate oligopaint probes, as users are able create fully designed libraries of tens of thousands of specific, single-stranded oligonucleotide sequences for binding particular genomic regions. Several researchers have demonstrated the effectiveness of OligoMix in their FISH-assays and have provided model strategies for generating oligopaint probes through their work. The strategies they present are important because they provide an experimental model other individuals can emulate and apply to new areas of fluorescence hybridization.

Oligopaint FISH Probes

PCR with Nick Endonuclease

The basic protocol for generation of the oligopaint FISH probes begins with a complex ssDNA library of thousands to hundreds of thousands of unique oligos; like the OligoMix libraries which are quickly and inexpensively generated using LC Sciences’ microfluidic array.

In one strategy, oligos can be designed with a pair of primer sequences (a forward primer and a reverse compliment of the reverse primer) that flank a genomic sequence bound on either side by sites for nicking endonucleases.

Incorporating two nicking endonuclease sites allows for the production of strand specific probes. Amplification with a labeled F primer and digestion yields a probe targeting the reverse complement of the genomic sequence.

Circle-to-Circle Amplification

In a second strategy, circle-to-circle amplification (c2ca) is used instead of PCR, to generate oligopaint probes. In circle-to-circle amplification, targeted template strands are cyclized via ligation into circular template strands, which are subsequently synthesized into chain-like repeated copies of the circular template by an enzyme with high processivity and strand displacement capacity.

This amplification method is unique, because it overcomes some of the drawbacks of PCR like sequence-dependent amplification bias. In addition to this, c2ca is an isothermal process and therefore does not require quick temperature changes which impede the scalability of PCR reactions.

Because c2ca amplified oligos do not carry a direct label, a common binding site is used for a fluorophore-labelled ‘secondary’ oligonucleotide.