Blocking Nucleic Acid Buffer for Hybridization Specificity
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Solution Overview
Problem
Current methods for detecting target nucleic acids using blocking agents are inadequate in preventing non-specific hybridization, especially when there are many non-target nucleic acid molecules, and existing blocking agents like surfactants and proteins lack accuracy in recognizing nucleotide sequences.
Innovation Solution
A buffer composition containing a blocking nucleic acid with a nucleotide sequence complementary to non-detection target nucleotides in non-target nucleic acids, used in concentrations 1 to 5 times higher than the target nucleic acid concentration, to suppress non-specific hybridization and enhance specific hybridization with nucleic acid probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional blocking agents (surfactants or proteins) are used to suppress non-specific hybridization, then the blocking effect is provided, but the accuracy in recognizing nucleotide sequences is insufficient
Solution Approach 1:
The invention changes the chemical nature of the blocking agent from non-nucleic acid components (surfactants, proteins) to nucleic acid components with specific sequences. This parameter change enables the blocking agent to recognize nucleotide sequences specifically through complementary base pairing, thereby improving both the blocking effect and sequence recognition accuracy simultaneously.
Solution Approach 2:
The invention introduces a specifically designed blocking nucleic acid sequence that acts as an intermediary between the probe and non-target nucleic acids. This blocking sequence hybridizes with non-target nucleic acids to prevent non-specific binding, while the concentration ratio control ensures it does not interfere with target detection.
2Reliability
If blocking nucleic acid with sequence complementary to non-target nucleic acid is used, then non-specific hybridization is suppressed, but the concentration ratio between blocking nucleic acid and target nucleic acid must be precisely controlled
Solution Approach 1:
The invention establishes specific parameter ranges for the concentration ratio of blocking nucleic acid to target nucleic acid (0.01-10 times, preferably 0.1-5 times). By defining these quantitative parameters, the invention transforms the complex concentration control requirement into a manageable specification that ensures effective blocking while minimizing interference with target detection.
3Reliability
If blocking agent concentration is increased to improve blocking effect, then non-specific hybridization suppression is enhanced, but the risk of interfering with target nucleic acid detection increases
Solution Approach 1:
The invention optimizes the concentration ratio parameter of blocking nucleic acid to target nucleic acid within specific ranges (0.01-10 times, preferably 0.1-5 times). This parameter optimization ensures sufficient blocking effect while preventing interference with target detection, resolving the contradiction between blocking effectiveness and detection accuracy.
Solution Approach 2:
The blocking nucleic acid is designed with specific sequence characteristics that provide local specificity - it has complementarity to non-target sequences but not to target sequences. This local quality difference allows the blocking agent to selectively bind non-target nucleic acids without interfering with target detection, even at elevated concentrations.
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 proposed buffer composition effectively suppresses non-specific hybridization of non-target nucleic acids with probes, significantly improving the detection efficiency of target nucleic acids by ensuring specific hybridization, even in low concentrations or when non-target nucleic acids differ by only one nucleotide.
Implementation Method 1
a buffer composition for hybridization, which comprises a blocking nucleic acid having a nucleotide sequence complementary to a region comprising at least a non-detection target nucleotide in a non-target nucleic acid
Implementation Method 2
a blocking agent that suppresses non-specific hybridization of a nucleic acid probe with a nucleic acid molecule other than the measurement target
Data Source
AI summary
In the case of using a blocking nucleic acid to prevent non-specific hybridization of a target nucleic acid with a nucleic acid probe, further excellent efficiency of detecting the target nucleic acid is achieved. A buffer composition used in hybridization of a target nucleic acid with a nucleic acid probe, wherein the buffer composition for hybridization contains a blocking nucleic acid comprising a nucleotide sequence complementary to a region comprising at least a non-detection target nucleotide in a non-target nucleic acid, in a concentration of one or more times higher than the concentration of a nucleic acid in a nucleic acid mixture consisting of the target nucleic acid and the non-target nucleic acid.