Low-Input DNA Labeling Composition for Comparative Genomic Hybridization

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Solution Overview

Problem

Existing methods for array-based comparative genomic hybridization (aCGH) face challenges in producing high-quality labeled DNA efficiently from low amounts of input DNA, such as 50-250 nanograms, which is crucial for detecting chromosomal aberrations in genetic diseases and cancers.

Innovation Solution

A method involving the use of random DNA primers, DNA-dependent DNA polymerase, and fluorescently labeled dUTP in the absence of metal chelating compounds like EDTA, with specific nucleotide concentrations and incubation conditions, to produce high-quality labeled DNA from low input amounts.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If conventional Nick translation or randomly primed DNA extension methods are used, then labeled DNA can be produced for aCGH, but the quality and quantity of labeled DNA are insufficient when starting with low input DNA amounts (50-250 nanograms)

Engineering Contradiction:

Improvequantity of labeled DNAVSAvoidquality of labeled DNA

Core Design Contradiction:

Quantity of substanceVSManufacturing precision

Solution Approach 1:

The invention changes the chemical parameters of the reaction system by eliminating metal chelating compounds (EDTA, EGTA) from the reaction buffer and using a simplified buffer composition with specific salt concentrations (e.g., 50-200 mM NaCl, 10-50 mM Tris-HCl). This parameter change enables efficient DNA extension and labeling from low input amounts while maintaining high product quality

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention extracts and removes metal chelating compounds (EDTA, EGTA) from the reaction system. By taking out these compounds that interfere with the DNA extension reaction, the method enables efficient incorporation of fluorescently labeled dUTP while maintaining reaction fidelity and producing high-quality labeled DNA from limited input material

Inventive Principle:

Principle #2Taking out (Extraction)

2Stability of the object's composition

If metal chelating compounds like EDTA or EGTA are present in the reaction buffer, then DNA stability may be maintained, but DNA extension and labeling efficiency are significantly reduced

Engineering Contradiction:

ImproveDNA stabilityVSAvoidDNA extension and labeling efficiency

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The invention converts the harmful effect of metal chelating compounds by completely removing them from the reaction system. This elimination allows the DNA extension reaction to proceed efficiently with high productivity while alternative measures (optimized buffer composition, controlled incubation conditions) maintain DNA stability throughout the process

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Productivity

If high concentrations of fluorescently labeled dUTP are used, then labeling efficiency increases, but the cost and complexity of the reaction system increase

Engineering Contradiction:

Improvelabeling efficiencyVSAvoidreaction system complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The invention optimizes the concentration parameters of fluorescently labeled dUTP in the reaction mixture, using concentrations that achieve high labeling efficiency (sufficient signal for aCGH) while maintaining a simple reaction system. The optimized concentration range ensures adequate signal strength without requiring excessive amounts of expensive labeled nucleotides or complex reaction conditions

Inventive Principle:

Principle #35Parameter changes

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 method enables the production of sufficient quantities of high-quality labeled DNA for aCGH, allowing for effective detection of chromosomal abnormalities with improved sensitivity and accuracy.

Implementation Method 1

a DNA-dependent DNA polymerase, such as a Klenow fragment

Methodology Applied

Scientific EffectDNA-dependent DNA polymerase catalysis: Enzyme

Implementation Method 2

to extend the random DNA primers on the genomic DNA, thereby forming fluorescently labeled DNA extension products

Methodology Applied

Scientific EffectPrimer extension:

Implementation Method 3

cooling the admixture to permit the random DNA primers to anneal to the genomic DNA in the admixture

Methodology Applied

Scientific EffectDNA annealing:

Implementation Method 4

heating the admixture to denature the genomic DNA

Methodology Applied

Scientific EffectDNA denaturation:

Data Source

PatentUS12442031B1DNA labeling compositions and methods for comparative genomic hybridization

Publication Date: 2025.10.14 ENZO BIOCHEM INC

AI summary

The invention provides compositions and methods for preparing high-quality labeled DNA for use in array-based comparative genomic hybridization in amounts sufficient therefor starting with a low amount of input genomic DNA, such as 50-250 nanograms input genomic DNA.