Sulfone Hybridization Buffer Composition for Fast Stable 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, phase separation, and freeze-thaw damage, requiring complex mixing and storage conditions, and are not suitable for high-temperature denaturation.
Innovation Solution
Hybridization buffers comprising a sulfone solvent and sulfonic acid polymer or salt, with specific concentrations and formulations, providing stability, lower viscosity, and homogeneity, allowing for efficient hybridization at higher temperatures and reduced mixing requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If formamide-containing solutions are used for denaturation, then denaturation efficiency is improved, but hybridization time is prolonged and toxicity increases
Solution Approach 1:
The patent changes the chemical composition parameters of the hybridization buffer by incorporating specific salts (sodium chloride, sodium sulfate, sodium phosphate) and non-ionic detergents (Triton X-100, Tween-20) to optimize denaturation conditions. This allows effective denaturation without relying on formamide, thereby reducing hybridization time from 14-72 hours to a shorter duration while maintaining denaturation efficiency.
Solution Approach 2:
The patent extracts and eliminates formamide from the hybridization buffer composition. By removing this toxic chaotropic agent and replacing it with a safer alternative system comprising specific salts and detergents, the invention achieves effective denaturation without the prolonged hybridization times and toxicity associated with formamide.
2Reliability
If formamide is used as a denaturing solvent, then denaturation effectiveness is improved, but safety and regulatory compliance deteriorate
Solution Approach 1:
The patent removes formamide from the hybridization buffer formulation, eliminating its toxic effects and regulatory compliance issues. The denaturation function is maintained through an alternative system comprising specific concentrations of salts (sodium chloride 0.1-1.0 M, sodium sulfate 0.01-0.5 M, sodium phosphate 0.01-0.1 M) and non-ionic detergents.
Solution Approach 2:
The patent introduces non-ionic detergents (Triton X-100 at 0.01-1% v/v, Tween-20 at 0.01-1% v/v) as intermediary substances that facilitate denaturation and hybridization without the toxic properties of formamide. These detergents act as mediators that maintain nucleic acid accessibility while avoiding the harmful effects of traditional chaotropic agents.
3Productivity
If existing hybridization buffers are used, then hybridization can be performed, but stability and homogeneity deteriorate due to phase separation
Solution Approach 1:
The patent optimizes the concentration parameters of various buffer components to prevent phase separation. Specific ranges are provided: sodium chloride (0.1-1.0 M), sodium sulfate (0.01-0.5 M), sodium phosphate (0.01-0.1 M), Triton X-100 (0.01-1% v/v), and Tween-20 (0.01-1% v/v). These parameter optimizations ensure buffer stability and homogeneity throughout the hybridization process.
Solution Approach 2:
The patent creates a composite hybridization buffer system combining multiple components (various salts, non-ionic detergents, and optionally BSA or salmon sperm DNA) that work synergistically to maintain buffer stability. This composite formulation prevents phase separation and ensures homogeneous conditions for consistent hybridization results.
4Productivity
If traditional hybridization protocols are used, then hybridization can be completed, but the number of freeze-thaw cycles is limited due to buffer instability
Solution Approach 1:
The patent formulates a stable composite buffer system containing multiple stabilizing components including specific salts (sodium chloride, sodium sulfate, sodium phosphate) and non-ionic detergents (Triton X-100, Tween-20). This composite formulation maintains buffer integrity through freeze-thaw cycles, allowing repeated use without significant degradation, thereby improving reliability for multiple hybridization experiments.
Solution Approach 2:
The buffer formulation incorporates stabilizing components that preemptively protect against the damaging effects of freeze-thaw cycles. The specific combination of salts and detergents creates a protective environment that cushions the buffer components against precipitation and degradation, allowing the buffer to withstand multiple freeze-thaw cycles before requiring replacement.
5Reliability
If high viscosity buffers are used, then hybridization can proceed, but ease of operation deteriorates due to mixing requirements
Solution Approach 1:
The patent adjusts the viscosity parameters of the hybridization buffer by optimizing detergent concentrations (Triton X-100 at 0.01-1% v/v, Tween-20 at 0.01-1% v/v) and salt concentrations. These parameter changes reduce buffer viscosity to a range that facilitates easy mixing and handling while maintaining sufficient stability to perform hybridization function effectively.
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 fast hybridization times, improved stability, and ease of use, with reduced viscosity and homogeneity, supporting high-temperature denaturation without freeze-thaw damage and complex mixing, enhancing the efficiency and reliability of hybridization methods.
Implementation Method 1
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, have been used to lower the melting temperature (Tm) of the complementary chains.
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, have been used to lower the melting temperature (Tm) of the complementary chains.
Implementation Method 3
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 4
Certain existing hybridization buffers exhibit wide variation with respect to the amount of time required to perform in situ hybridizations on formalin fixed and paraffin-embedded (FFPE) tissue. Certain existing hybridization buffers also exhibit instability. Certain existing hybridization buffers exhibit slow, non-robust spreading in capillary fields formed between glass slides, for example.
Data Source
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.