Hairpin Formation via Force-Induced Strand Invasion
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Solution Overview
Problem
Current methods for preparing hairpins for single-molecule nucleic acid analysis are inefficient, especially when dealing with limited sample quantities, as they require DNA enzymes, result in sample loss, and have suboptimal hairpin-to-bead ratios, leading to incomplete analysis and loss of quantitative accuracy.
Innovation Solution
A method for constructing hairpin structures without DNA enzymes, where nucleic acid hairpins are assembled in bulk prior to analysis, using synthetic DNA components and pre-formed receiving molecules bound to surfaces, allowing for optimal use of the nucleic acid of interest and minimizing sample loss.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If classic DNA library construction techniques with DNA ligase are used to prepare hairpins, then hairpin structures can be formed, but sample loss occurs and quantitative accuracy is reduced
Solution Approach 1:
The invention extracts and removes the DNA ligase enzyme step from the traditional hairpin preparation process. By using pre-formed receiving molecules with complementary single-stranded regions that can directly hybridize to the nucleic acid of interest, the method eliminates the need for enzymatic ligation, thereby preventing sample loss while maintaining ease of manufacture.
Solution Approach 2:
The invention uses synthetic receiving molecules as templates or copies that contain the necessary structural elements (single-stranded regions A and A') to guide the formation of hairpins without requiring the original DNA template to be physically manipulated by enzymes. This copying approach preserves the quantitative integrity of the sample.
2Reliability
If hairpin-to-bead ratio is increased to ensure surface attachment, then more beads bind to surface, but multiple hairpin attachments per bead occur preventing freedom of movement
Solution Approach 1:
The invention applies local quality by designing receiving molecules with specific localized single-stranded regions (A and A') that are positioned to interact with specific regions of the nucleic acid of interest. This localized interaction ensures that each bead attaches to the surface through exactly one hairpin structure, maintaining both reliable attachment and freedom of movement for denaturation.
Solution Approach 2:
The invention replaces the mechanical/empirical optimization of hairpin-to-bead ratios with a molecular design solution. By engineering the receiving molecules with specific complementary sequences and structures, the system achieves optimal attachment stoichiometry at the molecular level, eliminating the need for high ratios and preventing multiple attachments while ensuring reliable surface binding.
3Quantity of substance
If hairpin preparation is performed with limited nucleic acid samples, then analysis can proceed, but quantitative accuracy and completeness of analysis are compromised
Solution Approach 1:
The receiving molecules are designed to self-assemble with the nucleic acid of interest through complementary base pairing of single-stranded regions. This self-service mechanism eliminates the need for enzymatic processing that would consume or lose sample material, allowing complete quantitative analysis of limited nucleic acid samples while maintaining measurement precision.
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
This approach enables the preparation of hairpins suitable for single-molecule analysis with minimal sample loss and improved quantitative accuracy, even with low-concentration samples, by forming hairpins directly without enzymatic steps and optimizing the hairpin-to-bead ratio.
Implementation Method 1
wherein said single-stranded region of sequence C is capable of hybridizing to a complementary single-stranded region C' of said HP2
Implementation Method 2
c) denaturing said HP1 in the presence of said HP2, wherein denaturing said HP1 comprises applying a force to said HP1 that induces separation of said single-stranded regions A and A' from said HP2
Data Source
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AI summary
The present invention relates to a method of preparation of substrates for nucleic acid sequencing reactions. More specifically, the present invention provides a new method of preparing hairpins using force-induced strand invasion. Hairpins prepared by this method and methods of nucleic acid analysis using these hairpins are also part of the present invention.