Double-Stranded Nucleic Acid Synthesis Without Solid Supports
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Solution Overview
Problem
Current methods for synthesizing double-stranded nucleic acids require solid supports and polymerase amplification, making them inefficient and difficult to automate, with high error rates and the need for additional purification steps.
Innovation Solution
A method involving the assembly of single-stranded oligonucleotides into dumbbell-shaped DNA molecules, which are then converted into linear double-stranded nucleic acids without the need for solid supports or polymerase amplification, using a series of enzymatic steps to eliminate loops and unassembled oligonucleotides, allowing for error correction and automated production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If solid support and polymerase amplification are used in current methods, then double-stranded nucleic acid can be synthesized, but the process becomes inefficient and difficult to automate
Solution Approach 1:
The invention extracts and removes the solid support step from the synthesis process. By performing ligation and assembly reactions in solution phase rather than on solid supports, the method eliminates the need for solid support attachment and removal steps, thereby improving efficiency and enabling automation.
Solution Approach 2:
The invention applies preliminary action by pre-designing oligonucleotide sequences with specific features (such as overhangs and complementary regions) that enable self-assembly and directional ligation. This pre-programming of assembly instructions allows the system to automatically generate the correct double-stranded structure without manual intervention.
2Manufacturing precision
If gel purification and affinity purification are performed, then pure double-stranded nucleic acid is obtained, but the process becomes more complex and time-consuming
Solution Approach 1:
The invention applies self-service through self-cleaving ribozymes or toehold-mediated strand displacement mechanisms that automatically remove unwanted single-stranded oligonucleotides and hairpin structures. The system purifies itself by using built-in molecular mechanisms to eliminate impurities without requiring external purification equipment or procedures.
Solution Approach 2:
The invention changes the physical-chemical parameters of the reaction system to favor the formation and stabilization of double-stranded nucleic acid while preventing the persistence of single-stranded intermediates. By controlling temperature, salt concentration, and pH, the method achieves high purity products directly from the reaction mixture.
3Length of moving object
If successive annealing and ligation processes are used on magnetic beads, then long double-stranded oligonucleotide can be synthesized, but further gel purification and affinity purification are needed
Solution Approach 1:
The invention extracts the magnetic bead solid support from the synthesis process and performs all assembly operations in solution. This eliminates the need for bead-based purification steps while maintaining the ability to synthesize long oligonucleotides through sequential ligation of shorter fragments.
Solution Approach 2:
The method employs self-cleaving mechanisms and selective stabilization that automatically differentiate between correctly assembled long double-stranded oligonucleotides and incomplete or incorrect products. The system self-purifies by making only the correct full-length products stable and recoverable.
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 method enables cost-effective and time-efficient production of double-stranded nucleic acids with low error rates, eliminating the need for gel purification and solid supports, and allowing for automated processing.
Implementation Method 1
reacting said plurality of single-stranded oligonucleotides and the first and second hairpin-forming oligonucleotides with a ligase to form a closed double-stranded nucleic acid
Implementation Method 2
the sequence of each single-stranded oligonucleotide being at least partially complementary with the sequence of at least one other single-stranded oligonucleotide of said plurality, and/or with the sequence of the single-stranded overhang of the first or second hairpin-forming oligonucleotide
Data Source
AI summary
The present invention relates to a method for generating a double-stranded nucleic acid having a predetermined sequence and a method for producing a double-stranded polynucleotide of interest. The invention further relates to a kit for the implementation of said methods.