Ligase Chain Assembly of DNA Molecules
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Solution Overview
Problem
Current DNA synthesis methods are inefficient and prone to errors, requiring error correction enzymes and being less suitable for automated synthesis due to reliance on solid-phase devices and time-consuming processes.
Innovation Solution
The method involves performing a ligase chain reaction with short oligonucleotides of 8-22 nucleotides in length, followed by polymerase chain assembly and PCR, to assemble DNA molecules with high sequence fidelity without the need for error correction enzymes, allowing for automated synthesis and high yields of high-fidelity DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis of starting oligonucleotides is used, then DNA assembly can be performed, but error rates increase and sequence fidelity decreases
Solution Approach 1:
The patent extracts and removes the harmful element (error correction enzymes) from the DNA assembly process by using a specialized ligase chain reaction that inherently prevents error propagation through overlapping oligonucleotide design and controlled extension conditions
Solution Approach 2:
The patent changes critical parameters including oligonucleotide length (5-20 nucleotides), overlap region length (2-5 nucleotides), and reaction temperature cycling to achieve high-fidelity assembly without error correction enzymes, transforming the assembly process from error-prone to high-precision
2Manufacturing precision
If error correction enzymes are used to reduce errors, then sequence fidelity improves, but process complexity and time increase
Solution Approach 1:
The patent removes error correction enzymes from the process entirely, replacing them with a ligase chain reaction system that achieves high fidelity through overlapping oligonucleotide assembly and controlled enzymatic ligation, thereby simplifying the process while maintaining or improving sequence fidelity
Solution Approach 2:
The DNA assembly system performs self-correction through the overlapping oligonucleotide design where multiple overlapping sequences compete to form the correct assembly, with the ligase enzyme selectively joining correct matches while mismatched oligonucleotides remain unligated and can be removed in subsequent washes
3Ease of manufacture
If solid phase devices are used for DNA synthesis, then assembly can be performed, but automation suitability decreases and time consumption increases
Solution Approach 1:
The patent replaces solid phase mechanical devices with a solution-based ligase chain reaction system that uses liquid-phase enzymatic ligation of overlapping oligonucleotides, enabling simpler automation through standard liquid handling robots and thermocyclers while maintaining high assembly efficiency
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 achieves error rates of fewer than 1 error per 2000 base pairs, eliminating the need for error correction enzymes and enabling efficient, automated synthesis of DNA molecules with high fidelity.
Implementation Method 1
contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides
Implementation Method 2
The set of polynucleotides are optionally contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly
Implementation Method 3
Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality
Data Source
AI summary
The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.