Probe and Target Denaturation Without Formamide in Hybridization

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Solution Overview

Problem

Traditional hybridization methods rely on formamide for denaturation, which is toxic, hazardous, and prolongs renaturation time, causing morphological destruction and high background signals.

Innovation Solution

The use of aqueous compositions with polar aprotic solvents for separate denaturation of probes and targets, reducing or eliminating formamide, and optimizing denaturation and renaturation conditions to enhance efficiency and safety.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If formamide is used for denaturation, then denaturation effectiveness is improved, but renaturation time is prolonged and morphology is destroyed

Engineering Contradiction:

Improvedenaturation effectivenessVSAvoidrenaturation time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The invention changes the chemical composition parameters of the denaturation solution by replacing formamide with alternative agents such as urea, thiocyanate, or combinations thereof. This parameter change allows effective denaturation to be achieved without the adverse renaturation delay caused by formamide, thus resolving the contradiction between denaturation effectiveness and renaturation time.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If formamide is used for denaturation, then denaturation effectiveness is improved, but sample morphology is destroyed

Engineering Contradiction:

Improvedenaturation effectivenessVSAvoidsample morphology

Core Design Contradiction:

ReliabilityVSShape

Solution Approach 1:

The invention modifies the chemical parameters of the denaturation environment by substituting formamide with gentler alternatives like urea or thiocyanate-based solutions. These alternative compositions achieve the necessary strand separation while being less disruptive to the structural integrity and morphological preservation of the sample, thereby resolving the contradiction between denaturation effectiveness and morphology preservation.

Inventive Principle:

Principle #35Parameter changes

3Reliability

If formamide is used for denaturation, then denaturation effectiveness is improved, but background signal increases

Engineering Contradiction:

Improvedenaturation effectivenessVSAvoidbackground signal

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The invention changes the chemical composition of the denaturation solution from formamide to alternative agents such as urea or thiocyanate. This parameter change eliminates the formamidation side reaction that causes high background signals, while still achieving effective denaturation, thus resolving the contradiction between denaturation effectiveness and background signal levels.

Inventive Principle:

Principle #35Parameter changes

4Reliability

If formamide is used for denaturation, then denaturation effectiveness is improved, but toxicity and hazard increase

Engineering Contradiction:

Improvedenaturation effectivenessVSAvoidtoxicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention modifies the toxicological parameters of the denaturation solution by replacing the highly toxic and regulated formamide with less hazardous alternatives such as urea or thiocyanate compounds. This substitution maintains denaturation effectiveness while significantly reducing toxicity and regulatory burden, thereby resolving the contradiction between denaturation effectiveness and safety.

Inventive Principle:

Principle #35Parameter changes

5Shape

If separate denaturation is used, then morphology preservation is improved, but process complexity increases

Engineering Contradiction:

Improvemorphology preservationVSAvoidprocess steps

Core Design Contradiction:

ShapeVSDevice complexity

Solution Approach 1:

The invention combines the separate denaturation steps into a unified process by using denaturation compositions that can simultaneously or sequentially treat both probe and target without requiring formamide. The use of alternative agents like urea or thiocyanate allows the separate denaturation protocol to be implemented with reduced procedural complexity and without the need for extensive formamide handling infrastructure, thus resolving the contradiction between morphology preservation and process complexity.

Inventive Principle:

Principle #5Merging (Combining)

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 lower background signals, preserved sample morphology, easier automation, and faster hybridization processes while minimizing toxicity and regulatory concerns.

Implementation Method 1

Formamide disrupts base pairing by displacing loosely and uniformly bound hydrate molecules, and by causing 'formamidation' of the Watson-Crick binding sites. Thus, formamide has a destabilizing effect on double stranded nucleic acids and analogs.

Methodology Applied

Scientific EffectHydrogen bonding disruption:

Implementation Method 2

This double helix structure is stabilized by hydrogen bonding between bases on opposite strands when bases are paired in a particular way (A+T/U or G+C) and hydrophobic bonding among the stacked bases.

Methodology Applied

Scientific EffectHydrophobic bonding disruption:

Implementation Method 3

Complementary base paring (hybridization) is central to all processes involving nucleic acid.

Methodology Applied

Scientific EffectComplementary base pairing:

Data Source

PatentUS12534755B2Compositions and methods for performing hybridizations with separate denaturation of the sample and probe

Publication Date: 2026.01.27 AGILENT TECHNOLOGIES INC

AI summary

The invention provides methods and compositions for separately denaturing a probe and target in hybridization applications. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences.