Hybridization Buffer Composition for Fast, Formamide-Free ISH
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Solution Overview
Problem
Existing hybridization buffers face challenges such as high toxicity due to formamide use, prolonged hybridization times, instability at low temperatures, and inhomogeneity, requiring complex handling and storage conditions, which affect the efficiency and safety of nucleic acid hybridization assays.
Innovation Solution
Hybridization buffers comprising sulfone solvents like sulfolane and sulfonic acid polymers or their salts, which provide stability, lower viscosity, and improved homogeneity, allowing for shorter hybridization times and easier handling, even at higher temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If formamide is used as a denaturing solvent in hybridization buffers, then denaturation efficiency is improved, but toxicity increases and hybridization time is prolonged
Solution Approach 1:
The patent removes formamide from the hybridization buffer composition entirely, extracting the harmful denaturing agent from the system. The invention achieves denaturation without formamide by using alternative chemical compositions that eliminate toxicity while maintaining or improving denaturation efficiency.
Solution Approach 2:
The patent changes the chemical parameters of the hybridization buffer by substituting formamide with alternative compounds. This parameter change eliminates the toxicological properties associated with formamide while preserving the functional ability to denature nucleic acids and enable hybridization.
2Temperature
If formamide is used to lower melting temperature, then hybridization conditions are improved, but hybridization time is prolonged
Solution Approach 1:
The patent changes the chemical composition parameters of the hybridization buffer to achieve different thermal properties. By using alternative chemicals instead of formamide, the buffer achieves effective hybridization at improved time scales while maintaining appropriate melting temperature characteristics for probe-target binding.
3Productivity
If certain hybridization buffers are used to reduce incubation time, then productivity is improved, but stability and homogeneity deteriorate
Solution Approach 1:
The patent uses a composite buffer composition containing multiple chemical components working together. This composite formulation achieves fast hybridization kinetics while maintaining excellent stability and homogeneity throughout the buffer, resolving the trade-off between speed and compositional stability.
Solution Approach 2:
The patent optimizes the chemical parameters of the buffer composition to achieve a balance where fast hybridization occurs without compromising stability. The specific chemical ratios and concentrations are tuned to provide both rapid productivity and long-term compositional stability.
4Stability of the object's composition
If hybridization buffers require storage at low temperatures, then stability is improved, but ease of operation deteriorates due to complex handling requirements
Solution Approach 1:
The patent changes the stability parameters of the buffer by using chemically stable alternatives to formamide-based compositions. This allows the buffer to be stored at higher temperatures without degradation, eliminating the need for complex cold chain storage and handling procedures.
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
The buffers enable efficient, stable, and homogeneous nucleic acid hybridization with reduced viscosity, facilitating faster hybridization processes and improved handling, while eliminating the need for complex storage and mixing procedures, thus enhancing safety and efficiency.
Implementation Method 1
a denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions
Implementation Method 2
Chaotropic agents, such as formamide, guanidinium hydrogen, and urea, which interfere with the Watson-Crick binding sites of nucleic acid bases and thereby disturb the hydrogen bonds between complementary nucleic acid bases
Implementation Method 3
these agents prolong the hybridization time compared to hybridization in an aqueous solution without a chaotropic agent. However, the use of chaotropic agents lowers the Tm
Implementation Method 4
Hybridization is an important and well-known property of nucleic acids. The double helix structure of DNA is stabilized by hydrogen bonding between bases on opposite strands when bases are paired in one particular way (A+T or G+C)
Implementation Method 5
stabilized by hydrogen bonding between bases on opposite strands
Data Source
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AI summary
The present disclosure relates to hybridization buffers for use in hybridization, for example, for use in in situ hybridization (ISH). Hybridization buffers and compositions comprising a sulfone solvent and polyvinyl sulfonic acid or a salt thereof, and methods of making and using, the same are disclosed.