PEG-Mediated Nucleic Acid Assembly Single-Step Method
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Solution Overview
Problem
Current nucleic acid assembly methods require multiple steps, are inefficient for larger DNA fragments, and struggle with assembling genes rich in AT or GC sequences, often necessitating separate pre-assembly and PCR amplification steps.
Innovation Solution
A single-step method involving overlapping oligonucleotides, DNA polymerase, dNTPs, and a crowding agent like PEG, which facilitates nucleic acid assembly through denaturation, annealing, and extension phases, allowing for the assembly of longer DNA fragments and challenging sequences in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional two-step assembly methods are used, then assembly can be performed with standard techniques, but the process requires separate pre-assembly and PCR amplification steps increasing time and complexity
Solution Approach 1:
The patent combines the pre-assembly and PCR amplification steps into a single unified reaction. The assembly mixture contains all necessary components (oligonucleotides, DNA polymerase, dNTPs, PEG) to perform both assembly and amplification simultaneously, eliminating the need for separate steps and reducing overall process time.
Solution Approach 2:
The assembly mixture serves multiple functions: it acts as both the pre-assembly reaction medium and the PCR amplification buffer. The same mixture performs oligonucleotide assembly, product amplification, and maintains optimal conditions for DNA polymerase activity throughout the process.
2Length of stationary object
If DNA polymerase is used to build longer DNA fragments during assembly, then amplification can occur, but the method becomes inefficient for larger DNA fragments
Solution Approach 1:
The patent optimizes reaction parameters including PEG concentration (2-10% w/v), oligonucleotide concentration (50-200 nM), and DNA polymerase amount to enable efficient assembly and amplification of large DNA fragments. The extended extension phase (5-15 minutes) allows complete synthesis of long fragments.
Solution Approach 2:
The extension phase time is dynamically adjusted based on the expected size of the final DNA fragment. For fragments greater than 1 kb, the extension phase is extended to 5-15 minutes, and the number of cycles is optimized to balance assembly completeness with amplification efficiency.
3Adaptability or versatility
If standard assembly methods are used, then simple protocols can be followed, but the methods are unable to assemble nucleic acids having challenging nucleotide content
Solution Approach 1:
The patent adjusts the annealing temperature based on the melting temperature (Tm) of the oligonucleotides, which varies with sequence composition. For AT-rich or GC-rich sequences, the annealing temperature is optimized to ensure proper hybridization. The PEG concentration is also adjusted to facilitate assembly of challenging sequences.
Solution Approach 2:
The method uses multiple overlapping oligonucleotides that copy and verify each other's sequences through homologous recombination. This redundant copying mechanism ensures high fidelity assembly even for sequences with challenging nucleotide content, as errors in individual oligonucleotides can be corrected by matching sequences in other oligonucleotides.
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 significantly reduces the time and cost of assembling nucleic acids, enabling the construction of larger DNA fragments and genes with high AT or GC content, and eliminates the need for separate pre-assembly and amplification steps, achieving robust and efficient nucleic acid assembly.
Implementation Method 1
The presence of the crowding agent facilitates the nucleic acid assembly process of the invention. The crowding agent is polyethylene glycol (PEG).
Implementation Method 2
contacting a set of overlapping oligonucleotides with a DNA polymerase, a mixture of dNTPs, and a crowding agent to form an assembly mixture
Implementation Method 3
each cycle comprising a denaturation phase, an annealing phase, and an extension phase
Data Source
Figure 1A~1B
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Figure 3
AI summary
The present invention discloses methods for assembling a nucleic acid molecule from a set of overlapping oligonucleotides. The method involves contacting a set of overlapping oligonucleotides with a DNA polymerase, a mixture of dNTPs, and a crowding agent to form an assembly mixture. In one embodiment the crowding agent is polyethylene glycol (PEG). The presence of the crowding agent facilitates the nucleic acid assembly process of the invention. The assembly mixture is then subjected to multiple cycles, each cycle comprising an annealing phase, an extension phase, and a denaturation phase, and the desired nucleic acid molecule is thereby assembled. In some embodiments one or more of the phases are time varied.