Technology – KromaTiD

What is dGH?

KromaTiD’s directional Genomic Hybridization™ (dGH) technology combines bioinformatics driven design of unique single-stranded synthetic probes with strand-specific hybridization strategies, and is the only genomics approach capable of detecting DNA sequence, location and orientation in a single test.

dGH begins with bioinformatic analysis in order to identify probe target sequences.

Next, chromosomal directionality and repeat regions within the target sequence are identified.

The result is probes that are designed to hybridize solely to repeat free regions of the sequence of interest.

Using cutting-edge genomic and bioinformatic strategies, directional Genomic Hybridization can be efficiently utilized in projects ranging from one to thousands of samples.

With the ability to resolve very small inversions and translocations, dGH technology can be employed in both directed and de novo discovery studies, quickly providing data on new or variable rearrangements for your patient screening or target validation needs. If a complete genetic profile is required, pair dGH with NGS on the same samples to build a comprehensive mutation profile.

For inversions or translocations with known breakpoints, targeted dGH assays are an efficient method of screening large populations or libraries and can be multiplexed and automated to provide data on multiple aberrations in the same test.

How does it work?

Once repeat-free target sequences are identified and dGH probes are designed, the innovative chromosomal preparation technique enables the identification of previously undetectable structural rearrangements such as inversions.

The dGH mechanism for inversion detection is summarized below:

In addition to their unique ability to detect inversions, KromaTiD’s dGH probes can provide high resolution traditional FISH data. When used as Pinpoint FISH reagents with standard FISH methodologies and equipment, dGH probes maximize assay performance by improving kinetics, reducing background and eliminating the need for blocking DNA (COT).

How is it different?

The dGH platform differs from traditional FISH as a result of its unique chromosomal preparation technique. Analog nucleotide incorporation during the DNA replication phase of the cell cycle enables exonuclease daughter strand degradation that leaves dGH prepped chromosomes single-stranded.

Single-stranded chromosomes allow for unidirectional dGH probe binding which enables the detection of orientation information that traditional FISH assays cannot provide.

Double stranded metaphase chromosome with an inverted segment.

Inversion differentiated as a result of single-stranded chromosome and unidirectional probe binding after dGH preparation and probe hybridization.

The same inversion would not be visualized using traditional FISH methodology and probes. The double stranded DNA would result in probes binding to both strands of the metaphase chromosome, making it impossible to discern DNA orientation.

While the preparation conditions differ, dGH does not require additional laboratory equipment. dGH probes can be visualized using standard FISH equipment and fluorescence microscopes found in many labs, making visualization and analysis as simple as possible.

Image Library

With dGH, researchers can directly visualize all manner of genomic structural variants including translocations, sister-chromatid exchanges, and previously undetectable inversions.

The image library below provides examples of real dGH and Pinpoint FISH structural rearrangement detection imaging. Note that chromosomal aberrations are circled in white.

dGH Paint
This image provides an example of an assay using dGH chromatid paints to visualize inversions. The inversions, circled in white, are represented by fluorescent signal on the arm opposite to the painted side of the chromosome.
Targeted dGH
This image shows an example of a targeted dGH assay. As opposed to dGH paints, targeted dGH assays are designed to give strand specific structural data at a specific chromosomal region of interest. Normal chromosomes in this assay appear as a single pair of red and green dots. The aberrations circled here demonstrate a loss of genetic material in one chromosome (green dot without corresponding red dot) and a gain of genetic material in another chromosome (two red dots and a green dot).
Pinpoint FISH
This pictures demonstrates a metaphase Pinpoint FISH assay designed to monitor two genomic loci near the telomeric ends of a pair of homologous chromosomes. No chromosomal aberrations are visible in this picture, the green and red probes are not displaced from their expected positions.
dGH Paint
This image provides another example of an assay using dGH chromatid paints to visualize chromosomal abnormalities. The aberration shown here is demonstrated by the fluorescent signal on the dark arm of the circled chromosome.
Targeted dGH
This image shows an example of a targeted dGH assay designed to monitor specific genomic loci on two pairs of homologous chromosomes. Normal chromosomes are represented by one red and one green dot. Chromosomes with aberrations are circled and visualized by two red dots (gain of material) and no red dots (loss of material).
dGH Paint
Here, a reciprocal translocation and an inversion involving the painted chromosome and a random chromosome is demonstrated with a dGH paint assay. The circle on the painted chromosome highlights the loss of fluorescent signal on this chromosome. The corresponding fluorescence gain and an inversion within the translocated fragment on the random chromosome is highlighted with another circle.
dGH Paint
This image provides an example of an assay using dGH chromatid paints to visualize inversions. The inversions, circled in white, are represented by fluorescent signal on the arm opposite to the painted side of the chromosome.
dGH Paint
Example of a dGH probe ladder (pink) on a normal chromosome designed for the antisense strand and used in combination with chromatid paint (green) to narrow breakpoint region locations.
dGH Paint
Example of a dGH probe ladder (pink) and chromatid paint (green) with inverted regions circled.
Pinpoint FISH
This pictures demonstrates a Pinpoint FISH assay in interphase designed to monitor one pair of homologous genomic sites. A normal site is represented by the overlapping red and green probes resulting in a yellow color. A gene break-apart aberration is shown by the probe separation and visualized as the distinct red and green dots circled here.

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Pinpoint FISH
dGH Paint
dGH Paint
Targeted dGH
dGH Paint
Targeted dGH
dGH Paint
dGH Paint
dGH Paint
Pinpoint FISH

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Areas of application

The versatility of the dGH platform enables it to provide value in numerous areas of biomedical research. Learn more about dGH applications in a specific field by following one of the links below.

Gene Editing

Through structural analysis of baseline and edited cell populations, dGH is able to help optimize editing processes, quantify and identify on/off-target effects, and provide a crucial metric for quality control in editing trials and experiments.

Genetic Diseases

dGH has already helped identify previously undetected structural errors in patients. It enables clinicians and researchers to screen for rare chromosomal mutations and variants that other cytogenomic methods are unable to detect.

Oncology

dGH assays provide single cell analysis that reliably detects low-frequency, complex variations in heterogeneous cell populations, making it ideal for identifying potentially oncogenic mutations.

Radiation Biodosimetry

KromaTiD was founded on a grant from NASA which facilitated the development of dGH in order to track ionizing radiation induced DNA damage. dGH is capable of providing the foundation for multiple dose response assays including inversions, dicentrics and translocations.

Delivery

Cellular reagent delivery can result in unintended genomic consequences. dGH gives researchers a simple method for detecting and quantifying structural damage associated with experimental delivery conditions.

Although these applications represent the majority of work being done with dGH, this list is by no means comprehensive. Contact us today and we’ll provide a free consultation to determine if dGH can help with your project!

Assays and benefits

Quantitative Mutation Size and Location

Since dGH assays are based on a defined library of calibrated genomic probes, they can precisely locate and quantitatively size rearrangements.

Visual Orientation Data

When used on metaphase chromosomes, dGH is the only imaging technology capable of providing sequence, location and orientation information in a single assay.

The KromaTiD platform enables inversion detection with a resolution that is orders of magnitude greater than any competing technique.

Chromosome and Chromatid Assay Format

Using the same probes in different test conditions, KromaTiD assays can query entire chromosomes (double-stranded applications like FISH) or individual chromatids (single-stranded applications) in interphase or metaphase cells.

Unique Specificity

Our probes are designed to target unique genomic sequences, so KromaTiD assays require no blocking DNA (COT), exhibit no non-specific background, and demonstrate improved hybridization performance.

The Broadest Assay Range

In a single assay, KromaTiD products detect the broadest possible spectrum of chromosome rearrangements, including those assayable by standard FISH technologies (e.g. translocations between chromosomes) as well as intra-chromosomal rearrangements such as cryptic inversions.

Single Cell Analysis

dGH assays generate imaging data on a cell-by-cell basis so are ideal for determining mutation heterogeneity within mixed cell populations while simultaneously identifying recurrent rearrangements.

What is dGH?

How does it work?

How is it different?

Image Library

dGH Paint

Targeted dGH

Pinpoint FISH

dGH Paint

Targeted dGH

dGH Paint

dGH Paint

dGH Paint

dGH Paint

Pinpoint FISH

Pinpoint FISH

dGH Paint

dGH Paint

Targeted dGH

dGH Paint

Targeted dGH

dGH Paint

dGH Paint

dGH Paint

Pinpoint FISH

Areas of application

Gene Editing

Genetic Diseases

Oncology

Radiation Biodosimetry

Delivery

Assays and benefits

Quantitative Mutation Size and Location

Visual Orientation Data

Chromosome and Chromatid Assay Format

Unique Specificity

The Broadest Assay Range

Single Cell Analysis

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