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 dGH Works

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.

Why dGH Works

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).

dGH is 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.

dGH SCREEN

What is dGH SCREEN?

SCREEN Single-Cell Rearrangement Event Evaluation and Numbering

dGH SCREEN™ is a single cell assay designed to monitor structural variants throughout the genome in an entirely de novo fashion. By utilizing directional Genomic Hybridization technology combined with fluorescence labeling patterns and chromosomal aggregation strategies, dGH SCREEN provides the most comprehensive and high-resolution karyographic analysis available.

With dGH SCREEN, researchers are able to track and localize a wide variety of chromosomal rearrangements within a 10kb limit of detection:

  • Exchange Events including reciprocal, balanced and allelic translocations
  • Orientation Events including inversions, recombinations and sister chromatid exchanges
  • Chromosomal Gain & Loss Events including sister chromatid fusions, dicentrics/acentrics, fragmentation/chromothrypsis, polypoidy, aneuploidy, monosomy, polysomy

dgh-in-site-assays

Figure 1: Example of the first step in the dGH SCREEN analysis process. A metaphase spread is hybridized with 5 color dGH probes designed to measure known and unknown variants in order to provide a whole genome survey of structural rearrangements in a single cell.

SPIN OFF ARTICLE FROM NASA.GOV

SCREEN: Sort

Figure 2: Step 2 in dGH SCREEN analysis. Chromosomes are sorted by color and morphology to enable localization of rearrangement events.

APPLICATIONS

  • Monitoring Cellular Engineering Outcomes: Genome-wide, cell-by-cell and chromosome-by-chromosome assessment of structure, pre- and post modification
  • Orthogonal Data for Sequencing: Genome-wide, confirmatory data regarding rearrangements predicted with long read and other NGS analyses
  • Structural Integrity QC: A straightforward yardstick by which to measure the relative stability of cell lines, or to screen and compare candidate cell lines, based on total genomic structural variation metrics.
  • Variant Discovery: Discover previously unknown mutations by de novo assessment of single cells from patient sub-types
  • Genomic Stability Assessment: Track persistence of variants over time, passages, process variable changes, etc.

SCREEN: Karyograph

Example Data Output

Figure 3: Step 3 in dGH SCREEN analysis. Chromosomes are organized karyographically and aggregate sample rearrangement data is gathered for analysis of genomic stability and structural integrity.

HIGH RESOLUTION ANALYSIS

In addition to being an unbiased tool for the detection of structural variants throughout the genome,  dGH SCREEN offers high resolution analysis of DNA repair activity by monitoring hallmarks of genomic instability, including sister chromatid exchanges.

This analysis produces vast amounts of rearrangement data which can be used to create stability and structural variance profiles for complex and heterogeneous cell populations, making dGH SCREEN one of the most comprehensive and robust karyographic assays available.

For more information on how dGH SCREEN can transform your research, go to our dGH SCREEN product pageorcontact us here.

dGH In-Site

What is In-Site?

dGH in-Site™ Probes harness KromaTiD’s Directional Genomic Hybridization technology to provide whole genome tracking of inserted DNA cassettes as small as 2 kilobases. Unidirectional dGH probes provide a single cell, genome-wide perspective of cellular engineering outcomes, including tracking of viral and non-viral mediated insert integration (CRISPR/Cas and alternative systems).

in-Site - All Channels: Normal

Figure 1: Example of the dGH in-Site™ assay tracking on/off-target integration in an edited iPSC. Shown here in both homologs are two bracketing probes (green) on the centromeric and telomeric sides of the edit site. Insert probes (yellow) demonstrate on-target integration in one homolog (circled).

  • On- and off-target integration metrics
  • Clean integration data, even from complex or heterogeneous cell populations
  • A unique, whole genome, orthogonal method of direct visualization of inserts, without bioinformatic prediction of outcomes
  • Multi-channel fluorescence for flexible and multiplex assay design
  • Available for human, murine, canine, non-human primate and CHO cells.
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Channel 1: Fluorescence channels overlaid, insert and bracketing probes both visible on one copy of chromosome 22

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Channel 2: Green fluorescence channel, bracketing probes visible on both copies of chromosome 22

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Channel 3: Yellow fluorescence channel, insert probes visible on one copy of chromosome 22

in-Site All Channels: Off-Target Integration

Figure 2: Same dGH in-Site™ assay in an edited iPSC, demonstrating both on-target and random integration of insert sequence (yellow) throughout the genome.

As an orthogonal analysis to PCR/sequencing techniques, dGH in-Site™ assays enable, through direct visualization, definitive measurement of the presence or absence of inserts. This includes genome-wide distribution and orientation of transgenes or inserted segments. When used in combination with our targeted probes and/or dGH dosimetry paints, dGH in-Site™ assays can provide quantitative, single cell on- vs off-target outcomes of the delivery and editing process, and can be used to track the stability of the edited genome in both clonal isolates and non-clonal populations.

Offering the lowest limit of detection of integrated or genomic DNA targets by fluorescence, dGH in-Site is the most comprehensive tool available for researchers interested in tracking engineering outcomes in a de novo fashion.

For more information on how dGH in-Site can inform your research, you can download our information sheet here. or contact us here and we will reach out to you shortly!

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Channel 1: Fluorescence channels overlaid, insert and bracketing probes visible on one copy of chromosome 22 and off target inserts in multiple chromosomes

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Channel 2: Yellow fluorescence channel, insert probe signals on one copy of chromosome 22 (circled) and in multiple off-target chromosomes

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Channel 3: Green fluorescence channel, bracketing probe signals on both copies of chromosome 22

Pinpoint FISH

What is Pinpoint FISH?

Pinpoint FISH™ is a synthetic oligonucleotide based FISH assay designed to provide the highest resolution, lowest background, and lowest limit of detection available. By modifying the hybridization conditions and eliminating strand degradation steps, Pinpoint FISH™ allows researchers to expand beyond the capabilities of conventional FISH probes to detect smaller targets and design ultra-high specificity tests.

Our technology is fully compatible with your standard FISH equipment/conditions and BAC probes, and works with your established samples, workflow, and imaging system. Providing 3x the relative signal; 3x the resolution and 6x the signal-to-noise ratio; 50% lower background. Pinpoint probes can be designed for a wider range of targets and target sizes Pinpoint FISH assays can easily detect mutations with variable breakpoints free of repetitive sequences, and no blocking required. Pinpoint FISH PPF™ provides Robust performance and consistent hybridization quality.

kromatid assay

A lymphoblast cell line (GM15510) was fixative and probed with KromaTid p53 probe yellow.

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.

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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

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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.

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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.

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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.

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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.

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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.

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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.

Contact us

Contact us today for a free consultation to determine if dGH can help solve your research needs!

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