Crosslinked dsDNA Primer Labels for Multiplex Capillary Electrophoresis
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Solution Overview
Problem
Existing methods for genetic analysis using capillary electrophoresis are limited in detectable strand length and prone to non-specific binding due to challenges in synthesizing long primers and using labels that affect mobility and binding.
Innovation Solution
Utilizing interstrand crosslinked double-strand DNA molecules as primer labels, which can be easily synthesized and prevent non-specific binding, allowing for a wider range of detectable strand lengths and improved mobility differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If long primers (>100 bp) are used to extend detectable strand length zone, then the detectable strand length zone is extended, but it becomes difficult to chemically synthesize the primer and non-specific binding occurs during single base extension reaction
Solution Approach 1:
The primer is divided into two separate components: a long nucleic acid strand (>100 bp) that provides the extended detectable length, and a short primer sequence that specifically binds to the target. This segmentation allows the long strand to be synthesized as a separate entity while the short primer maintains its specific binding capability, resolving the contradiction between extended length and synthesis difficulty.
Solution Approach 2:
A double-stranded nucleic acid structure acts as an intermediary between the long nucleic acid strand and the short primer sequence. The long strand forms a double-stranded structure with a complementary strand, and the short primer binds to the target through this intermediary structure. This prevents the long strand from causing non-specific binding while maintaining the extended detectable length.
2Measurement precision
If conventional labels are used to change electrophoretic mobility, then mobility differences are achieved, but non-specific binding occurs during single base extension reaction
Solution Approach 1:
The double-stranded nucleic acid structure serves as an intermediary that separates the labeling function from the binding function. The long nucleic acid strand with its label provides the mobility difference, while the short primer sequence specifically binds to the target. The double-stranded structure prevents the labeled long strand from directly interacting with the target, eliminating non-specific binding while preserving mobility differences.
Solution Approach 2:
The labeling function is segmented from the binding function. The long nucleic acid strand carries the label that changes electrophoretic mobility, while the short primer sequence is responsible for specific binding to the target. This functional segmentation resolves the contradiction between achieving mobility differences and maintaining specific binding.
3Adaptability or versatility
If the range used for analysis is limited to up to 120 bp, then chemical synthesis is feasible, but the multiplex detection capability is restricted
Solution Approach 1:
The primer system is segmented into a long nucleic acid strand (>100 bp) that extends the detectable range and a short primer sequence that maintains synthesis feasibility. By dividing the functional requirements between these two components, the system achieves extended multiplex detection capability while keeping the synthesizable portion within feasible length limits.
Solution Approach 2:
The solution moves from a single-dimension approach (primer length) to a two-dimension approach (separate long strand and short primer components). The long strand provides extended length in one dimension while the short primer maintains synthesis feasibility in another dimension, enabling enhanced multiplex detection without exceeding synthesis capabilities.
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
Enables simultaneous detection of more target nucleic acids with high sensitivity by extending the detectable strand length zone in electrophoresis and preventing non-specific binding, enhancing the multiplex detection capability.
Implementation Method 1
a first oligonucleotide (1) and a second oligonucleotide (2), each having a single-strand sequence, are hybridized to form a double-strand nucleic acid, an interstrand crosslink is formed between the first oligonucleotide (1) and the second oligonucleotide (2)
Implementation Method 2
the resulting reactant to capillary electrophoresis for analysis
Data Source
AI summary
Provided are a method and a means for improving a single base extension reaction method using capillary electrophoresis. Specifically, the present invention relates to a method for detecting presence of a target nucleic acid in a sample and/or determining a base of the target nucleic acid, the method including: preparing a sample containing or suspected of containing a target nucleic acid; preparing a primer 200 containing a double-strand nucleic acid tag 204 with an interstrand crosslink 203 and a primer nucleic acid 205 which specifically binds to the target nucleic acid; performing a single base extension reaction with the primer using the target nucleic acid as a template; and subjecting the resulting reactant to capillary electrophoresis for analysis.