Repeat-Depleted DNA Probes via Duplex Specific Nuclease Digestion

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Solution Overview

Problem

Current methods for generating DNA probes are hindered by the presence of repetitive sequences, which reduce specificity and require the use of blocking DNA, increasing costs and complexity in applications like FISH and CGH.

Innovation Solution

The use of duplex specific nucleases to selectively digest and remove repetitive sequences from DNA probes, allowing for the production of repeat-depleted probes that can hybridize specifically without blocking DNA, thereby enhancing probe specificity and reducing production costs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If blocking DNA is used to prevent hybridization of repetitive sequences, then probe specificity is improved, but production cost increases

Engineering Contradiction:

Improveprobe specificityVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent extracts and removes repetitive sequences from the probe DNA through enzymatic digestion using duplex-specific nucleases. This eliminates the need for blocking DNA while maintaining probe specificity, directly resolving the contradiction between improving specificity and reducing cost.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of adding blocking DNA to prevent repetitive sequence hybridization, the invention inverts the approach by removing the repetitive sequences from the probe itself. This fundamental reversal eliminates the need for additional blocking agents and reduces production costs.

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If blocking DNA is used to prevent hybridization of repetitive sequences, then probe specificity is improved, but process complexity increases

Engineering Contradiction:

Improveprobe specificityVSAvoidprocess complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

By extracting repetitive sequences from the probe through enzymatic digestion, the invention simplifies the overall process. The probe preparation becomes a single-step digestion process rather than a multi-step process involving blocking DNA addition, hybridization, and washing, thereby reducing process complexity while maintaining specificity.

Inventive Principle:

Principle #2Taking out (Extraction)

3Reliability

If hydroxyapatite chromatography is used to remove repetitive sequences, then probe specificity is improved, but manufacturing ease deteriorates

Engineering Contradiction:

Improveprobe specificityVSAvoidprocess simplicity

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent replaces the mechanical chromatography system (hydroxyapatite columns, gradient elution) with a biochemical enzymatic digestion system. This substitution dramatically simplifies the manufacturing process, making it easier to perform while achieving the same goal of removing repetitive sequences and improving probe specificity.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Reliability

If cross-linking agents are used to prevent hybridization of repetitive sequences, then probe specificity is improved, but process time increases

Engineering Contradiction:

Improveprobe specificityVSAvoidprocess time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

By directly extracting and removing repetitive sequences through enzymatic digestion, the invention eliminates the need for time-consuming cross-linking processes. The digestion reaction can be completed in a single incubation step, significantly reducing the overall process time while maintaining probe specificity.

Inventive Principle:

Principle #2Taking out (Extraction)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach results in highly specific and cost-effective DNA probes that can accurately target unique sequences, improving the sensitivity and reliability of hybridization reactions, such as FISH and CGH, by eliminating non-specific binding and signal interference from repetitive sequences.

Implementation Method 1

Duplex specific nucleases are added to the reannealed DNA to allow digestion of the double stranded DNA

Methodology Applied

Scientific EffectEnzymatic digestion: Enzyme

Implementation Method 2

The single stranded DNA is allowed to reanneal to form double stranded DNA based upon sequence homology

Methodology Applied

Scientific EffectNucleic acid hybridization: Chemical Bonding

Data Source

PatentUS11015227B2Methods and compositions to generate unique sequence DNA probes, labeling of DNA probes and the use of these probes

Publication Date: 2021.05.25 MENARINI SILICON BIOSYSTEMS SPA

AI summary

The invention relates generally to the field of the identification of DNA sequences, genes or chromosomes. Methods and composition to obtain Unique Sequence DNA probes are provided. Compositions comprised of and double stranded DNA containing Unique Sequences from which the repetitive sequences are eliminated according to the method described in this invention. The invention also relates to the preservation of cells that have been identified after immunomagnetic selection and fluorescent labeling in order to further interrogate the cells of interest. Furthermore the invention relates to genetic analysis of cells that have been identified after immunomagnetic selection and fluorescent labeling.