Mutagenesis Method Using Rolling Circle Amplification
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Solution Overview
Problem
Current mutagenesis methods face challenges in creating large randomized gene libraries with low template background and high yield, often requiring template modifications and resulting in significant bottlenecks during DNA transformation.
Innovation Solution
A method involving primer extension and multiple-primed rolling circle amplification (RCA) to selectively amplify mutated nucleic acid molecules, using techniques such as uracil-DNA glycosylase treatment and restriction enzyme digestion to render the template strand unfavorable for amplification, thereby reducing wild-type template contamination and increasing mutagenesis efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional primer extension methods are used with wild type gene template, then mutagenesis can be performed, but significant amount of wild type template molecules remain in the final product reducing library quality
Solution Approach 1:
The invention extracts and removes the wild type template molecules from the reaction mixture through selective digestion. The template DNA is modified to contain uracil residues which are not present in the mutagenized strands, allowing selective removal of template using uracil-DNA glycosylase and subsequent excision repair enzymes, thereby eliminating wild type background from the final library
Solution Approach 2:
The invention changes the chemical parameter of the template DNA by incorporating uracil residues into the template strand. This parameter change enables selective recognition and removal of the template by uracil-specific enzymes, while the mutagenized strands lacking uracil remain intact for amplification and transformation
2Reliability
If ds(U)DNA template is used with additional nitrocellulose filtering steps, then template removal is improved, but ease of template preparation is compromised
Solution Approach 1:
The invention employs self-service by using enzymatic systems that automatically recognize and process the uracil-modified template. The uracil-DNA glycosylase and excision repair enzymes specifically target uracil-containing DNA, providing automatic template removal without requiring external filtering steps or manual intervention, thereby simplifying the overall process
3Reliability
If stop codons are introduced at mutagenesis sites to prevent wild type translation, then wild type protein translation is blocked, but library size is reduced due to lower mutagenesis efficiency
Solution Approach 1:
The invention converts the potential harm of uracil incorporation (which could be detrimental to DNA stability) into a beneficial feature for selective template removal. The uracil residues, while potentially harmful, enable precise discrimination between template and mutagenized strands through uracil-specific enzymatic processing, thereby achieving both wild type suppression and high library yield
4Productivity
If transformation of DNA to host cells is performed, then mutagenized DNA can be propagated, but this step becomes the biggest bottleneck in the gene library production line
Solution Approach 1:
The invention performs preliminary action by removing the wild type template and enriching the mutagenized strands before transformation. This pre-enrichment step ensures that only mutagenized DNA is transformed into host cells, eliminating the need for subsequent screening and reducing the transformation bottleneck by increasing the efficiency and yield of transformable DNA
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 high mutagenesis efficiency, with up to 100% functional mutagenesis efficiency and a significant increase in the yield of mutated clones, overcoming the limitations of traditional methods by minimizing template contamination and streamlining the process.
Implementation Method 1
rendering the template strand unfavourable for rolling circle amplification (RCA)
Implementation Method 2
amplifying the mutated strand by multiple-primed rolling circle amplification
Implementation Method 3
digesting remaining unmutated methylated ds(U)DNA template with DpnI
Implementation Method 4
carrying out a primer extension and ligation reaction to form a double-stranded heteroduplex
Data Source
AI summary
The present invention provides a mutagenesis method wherein a nucleic acid molecule is mutagenized with at least one mutagenesis primer in a primer extension reaction and subsequently amplified by rolling circle amplification (RCA). The method involves the step of rendering the template strand unfavorable for RCA. The method involves steps leading to selective amplification of only the mutated strand by a strand-displacing DNA polymerase. Multiple copies of the mutated plasmids are generated during multiple-primed RCA and the resulting DNA is transformed for use. The method is suitable for mutating both single-stranded and double-stranded DNA. The present invention also provides a kit for use in the mutagenesis method.