Ammonium-Based Hybridization Buffer for Multiplex Nucleotide Detection
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Solution Overview
Problem
Current hybridization buffers used in multiplex analyses, particularly those containing tetramethylammonium ions, are toxic and have an unpleasant odor, making them hazardous to handle and store, while also posing challenges in achieving optimal hybridization conditions due to their impact on the G-C base content of nucleotide sequences.
Innovation Solution
An aqueous hybridization and detection buffer comprising unsubstituted ammonium ions, a buffer substance to stabilize pH, a chelating agent, and a surfactant, which provides appropriate stringency and sensitivity without the toxicity or odor issues of tetramethylammonium-based buffers, allowing for efficient hybridization and detection of nucleotide sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If tetramethylammonium-based buffers are used for hybridization, then appropriate stringency and hybridization kinetics are achieved, but toxicity and unpleasant odor increase
Solution Approach 1:
The patent substitutes tetramethylammonium ions with unsubstituted ammonium ions, changing the chemical composition parameter while maintaining the ionic strength and concentration parameters necessary for hybridization stringency. This substitution eliminates the harmful properties (toxicity and odor) associated with tetramethylammonium compounds while preserving the functional performance of the buffer.
2Productivity
If ammonium ion concentration is increased to improve hybridization kinetics, then reaction rate increases, but signal-to-noise ratio may be affected
Solution Approach 1:
The patent optimizes the concentration parameter of ammonium ions to achieve an optimal balance between hybridization reaction rate and signal-to-noise ratio. By carefully controlling the ionic strength and ammonium ion concentration, the buffer enables fast hybridization kinetics while maintaining high detection precision and signal-to-noise ratio in multiplex analyses.
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 buffer enables selective detection of nucleotide sequences with improved sensitivity and signal-to-noise ratio, supports high-throughput analysis, and can be stored at room temperature for extended periods without degradation, while being safer to handle and transport.
Implementation Method 1
the salt concentration (or ionic strength) as well as the presence of organic solvents in the reaction solution and the pH value of the same, the stringency and also the kinetics of the hybridization process
Implementation Method 2
The hybridization and/or detection buffer according to the invention further preferably contains at least one chelating agent for complexing divalent metal ions
Implementation Method 3
The hybridization and/or detection buffer according to the invention contains 0.1 - 5%, preferably 0.4 - 2.5%, of the surfactant as 1 x buffer
Data Source
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AI summary
The present invention provides an aqueous hybridization and/or detection buffer for the selective detection of nucleotide sequences in a sample, as well as a method for the selective detection of one or more nucleotide sequences (target nucleotide sequences) in a sample. The buffer according to the invention is particularly suitable for the simultaneous detection of several target nucleotide sequences in a sample (so-called multiplex analysis) using solid-phase-bound oligonucleotide probes.