Template-Free Long-Chain Nucleic Acid Synthesis by Oligonucleotide Assembly
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Solution Overview
Problem
Existing DNA synthesis methods struggle with high error rates, limited length, and increased costs as sequence length increases, particularly for enzymatic assembly and chemical synthesis, making it difficult to achieve high accuracy and efficiency in synthesizing long-chain nucleic acids.
Innovation Solution
A template-free method combining enzymatic synthesis of double-stranded oligonucleotides and ligase assembly, involving specific ligation steps with elution to remove impurities, ensuring high accuracy and efficiency in synthesizing long-chain nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis method is used to produce long sequences, then high-throughput production is achieved, but error rate increases and accuracy decreases
Solution Approach 1:
The patent divides the synthesis process into two distinct stages: (1) chemical synthesis of short oligonucleotide segments (50-200 bp) with high accuracy, and (2) enzymatic assembly of these segments into long sequences (1-10 kb). This segmentation allows each stage to operate in its optimal performance range, maintaining high throughput while ensuring high accuracy through the use of accurate chemical synthesis for short segments and high-fidelity enzymatic assembly for combining them.
2Length of stationary object
If enzymatic assembly method is used to increase sequence length, then longer molecules are obtained, but accuracy and yield decrease
Solution Approach 1:
The patent performs preliminary chemical synthesis of short oligonucleotide segments with high accuracy before attempting enzymatic assembly. These pre-synthesized accurate segments serve as building blocks that are then assembled enzymatically. This preliminary action ensures that the input materials for enzymatic assembly are already highly accurate, thereby maintaining overall sequence fidelity even as chain length increases to 1-10 kb.
Solution Approach 2:
The patent optimizes enzymatic assembly parameters including using specific high-fidelity DNA polymerases and ligases, controlling reaction temperatures, adjusting nucleotide concentrations, and implementing multiple assembly cycles. These parameter changes enable the enzymatic process to maintain high accuracy (＞99%) while achieving long chain lengths that would be impossible through chemical synthesis alone.
3Adaptability or versatility
If de novo synthesis is used for long-chain nucleic acid, then template-free synthesis is achieved, but synthesis efficiency and accuracy cannot be guaranteed
Solution Approach 1:
The patent segments the template-free synthesis process into chemical synthesis of short oligonucleotides followed by enzymatic assembly. This segmentation allows the use of high-accuracy chemical synthesis for short segments while using enzymatic methods (which are more adaptable for long sequences) for assembly, thereby achieving both template-free capability and high accuracy for long-chain nucleic acids.
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 method achieves continuous synthesis from single nucleotides to long-chain nucleic acids with high accuracy, low complexity, and low cost, using common reagents and instruments without the need for templates.
Implementation Method 1
a DNA polymerase, under extension conditions, is used to extend a 3' end of the primer
Implementation Method 2
combination and ligation of the double-stranded oligonucleotides to obtain a target long-chain nucleic acid
Implementation Method 3
treating first composite sequences marked as N/2 with endonuclease to obtain first free composite sequences
Data Source
AI summary
The present application relates to the fields of molecular biology and biotechnology, and in particular to a method for template-free de novo synthesis of a long-chain nucleic acid, including the following steps: S1, synthesis of double-stranded oligonucleotides; and S2, combination and ligation of the double-stranded oligonucleotides to obtain a target long-chain nucleic acid. The present application achieves continuous synthesis from single nucleotides to long-chain nucleic acids by means of the combination of S1 and S2, and has the advantages of no need for templates, high accuracy, low complexity and low cost.