The size of the smallest rearrangement (inversion or translocation) detectable by dGH technology is assay-dependent. For example, the KromaTiD chromosome 3 chromatid painting assay is designed to routinely resolve any 1 Mb inversion; in special cases, this same assay can detect inversions as small as 2 kb. Some KromaTiD custom assays are designed to target smaller regions or genes and can routinely detect rearrangements as small as a few kb.
KromaTiD chromatid painting and gene-specific probes are currently available labeled with fluorescein, Cyanine Dye 3 or Cyanine Dye 5; assay results can be viewed with microscope filters commonly used with these fluorophores. Other fluorescent labels are available upon request.
Chromatid-specific probes have the flexibility of working in either single-stranded or double-stranded applications. While some dGH assays work optimally on cells that are alive and actively dividing, KromaTiD probes can be used as Pinpoint FISH probes in non-growing samples.
dGH assays fit well on either end of a typical sequencing workflow. When performed prior to sequencing, dGH chromatid painting assays are capable ofdiscovering and localizing previously unknown inversions, effectively decreasing the size and shortening the time ofthe follow-up sequencing project. When sequencing results indicate a potential inversion, KromaTiD can design a 2- or 4-probe assay to bracket the putative breakpoint(s); dGH assays conducted post-sequencing can be used to confirm the suspected inversion. When provided with actively dividing test cells that contain a suspected inversion, KromaTiD can perform the post-sequencing dGH assays as a contract research project, ideally in a blinded fashion along with a control cell line.
Yes. KromaTiD can design gene-specific or chromosome painting dGH assays for any genome that has been previously sequenced. Because of high degrees of homology, stock and custom KromaTiD human dGH assays perform well for great ape species.
Because dGH technology is so flexible, KromaTiD often designs custom assays to help interpret results from other platforms, including sequencing and CGH array systems. dGH assays are ideal for analyzing chromosomal structural rearrangements which typically cause aberrant results on whole genome platforms like aCGH and sequencing. dGH assays are also capable of detecting deletions.
While appropriate for detecting translocations, SKY and MFISH cannot be used to reliably detect or discover inversions. By contrast, dGH assays are capable of detecting all structural rearrangements (including inversions) in a single assay, without the need for specialized imaging systems-effectively lowering the cost per sample compared to SKY and MFISH.
Yes. KromaTiD offers a growing number of mini-painting assays and gene-specific assays as catalog products. We also provide custom dGH assays for the entire human genome; our clients define their desired target area and preferred probe coverage and KromaTiD then designs a custom dGH assay to target the specific region, gene or arm using one or more requested fluorescent tags.
Although dGH assays can be designed to target smaller areas, dGH probes usually target unique chromosomal regions of approximately 6 kb. To cover larger regions, dGH painting and mini-painting assays employ a large number of targeted probes to create a pattern of discrete “spots” all along the target sequence.
Any lab already equipped to perform FISH assays can run dGH assays with just a few minor modifications. From a sample type standpoint, dGH assays require metaphase preparations from cells which have been grown in BrdU and BrdC (both provided in KromaTiD kits) for one cell cycle. In addition to standard FISH equipment, dGH assays require a 365 nm UV light source (e.g. a UV Crosslinker) for performing incubations as well as Exonuclease III.
Absolutely. dGH assays are extremely flexible and can be designed to locate specific genetic features, including breakpoints.
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