Hybridization Compositions Using Reduced Formamide
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Solution Overview
Problem
Traditional nucleic acid hybridization methods, such as in situ hybridization, require aggressive conditions like high temperatures and high formamide concentrations, which can be destructive, toxic, and result in long processing times, leading to inefficient and morphologically damaging hybridization processes.
Innovation Solution
The use of an aqueous composition with reduced formamide concentrations (less than or equal to 25%) and controlled energy application, allowing for hybridization at lower temperatures or faster rates, using methods like heating and cooling, and potentially employing microwaves, to reduce the energy barrier for nucleic acid hybridization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If high temperatures and high formamide concentrations are used for denaturation, then nucleic acid strand separation is achieved, but nucleic acid structure is destroyed and processing time is prolonged
Solution Approach 1:
The patent changes the chemical parameters of the hybridization buffer by introducing alternative denaturants (guanidinium salts, betaine, formic acid) and adjusting buffer composition (SSC concentrations, detergent types) to achieve effective denaturation at lower temperatures, thereby reducing processing time while preserving nucleic acid structure integrity
Solution Approach 2:
The patent uses intermediary substances such as denaturant agents and buffer components that facilitate strand separation without requiring extreme temperatures, acting as mediators between the probe and target nucleic acids to enable hybridization under milder conditions
2Stability of the object's composition
If high formamide concentrations are used, then denaturation is facilitated, but toxicity increases and morphological structure is destroyed
Solution Approach 1:
The patent extracts and replaces formamide with alternative denaturants such as guanidinium thiocyanate, guanidinium hydrochloride, betaine, and formic acid, removing the toxic substance while maintaining the denaturation function through chemically equivalent or superior alternatives
Solution Approach 2:
The patent converts the harmful effects of high formamide concentrations into beneficial outcomes by using milder alternatives that achieve the same denaturation effect without toxicity, and potentially improves morphological preservation while maintaining nucleic acid strand separation capability
3Reliability
If high temperatures are used for hybridization, then probe binding is achieved, but energy consumption increases and nucleic acid bonds are broken
Solution Approach 1:
The patent changes the thermal and chemical parameters of the hybridization reaction by using alternative buffers and denaturants that enable reliable probe binding at lower temperatures, reducing energy consumption while maintaining or improving binding reliability through optimized buffer composition and probe design
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 enables faster hybridization times, reduces toxicity, and maintains the integrity of nucleic acid structures, achieving sensitive, flexible, and reliable hybridization procedures with shorter processing times, typically under 2 hours or less.
Implementation Method 1
causing 'formamidation' of the Watson-Crick binding sites
Implementation Method 2
displacing loosely and uniformly bound hydrate molecules
Implementation Method 3
stabilized by hydrogen bonding between bases on opposite strands when bases are paired in one particular way (A+T/U or G+C)
Implementation Method 4
hydrophobic bonding among the stacked bases
Implementation Method 5
aggressive conditions to disrupt the hydrogen and hydrophobic bonds in the double helix
Data Source
AI summary
The invention provides methods and compositions for hybridizing at least one molecule to a target. The composition comprises at least one nucleic acid sequence, formamide, and a hybridization solution, wherein the concentration of formamide is less than or equal to 25%.