Boolean OR-Type Variant Nucleotide Library Design
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Solution Overview
Problem
Existing methods for generating variant DNA libraries are limited by the vast size of sequence space, making it impractical to exhaustively test all possible sequences, especially when mutating multiple codons simultaneously, leading to libraries that are too large to be experimentally screened.
Innovation Solution
The use of Boolean OR-type mutations in PCR reaction mixtures with mutagenic primers allows for the creation of variant libraries where each variant has alternative mutations in specific target regions, reducing the overall library size significantly by employing asymmetric amplification and combining mutations in a combinatorial manner, enabling the mutation of multiple amino acids without testing all residues simultaneously.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional methods (error-prone PCR, DNA shuffling, site-directed mutagenesis) are used to generate variant libraries, then genetic diversity is created, but the library size becomes too large to be experimentally screened
Solution Approach 1:
The patent segments the sequence space by dividing it into multiple reduced libraries, each covering a specific subset of mutations. Instead of creating one exhaustive large library, the method creates several smaller libraries that collectively cover the entire sequence space, making experimental screening feasible while maintaining comprehensive genetic diversity coverage
Solution Approach 2:
The patent applies partial action by creating reduced libraries that cover only specific portions of the total sequence space in each experimental round. By iteratively applying the method across multiple rounds with different subsets, the complete sequence space is explored without requiring all variants to be present simultaneously in one library
2Adaptability or versatility
If the number of mutated codons is increased to explore more sequence space, then genetic diversity improves, but the library size becomes exponentially larger and impractical to screen
Solution Approach 1:
The patent divides the task of exploring sequence space into segments by creating multiple reduced libraries, each focusing on a specific subset of codons or mutation types. This segmentation allows comprehensive sequence space coverage to be achieved through iterative experimentation rather than requiring a single impractically large library
Solution Approach 2:
The patent adds the dimension of iteration to the library creation process. Instead of attempting to cover all sequence space in one dimension (single library), the method uses multiple dimensions (multiple iterative rounds with different reduced libraries) to achieve comprehensive coverage while maintaining manageable library sizes in each round
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 dramatically reduces the library size, allowing for the mutation of a greater number of positions while still testing all desired mutations, facilitating the search for improved variants within the vast sequence space, as demonstrated by significant reductions in library size and increased catalytic activity in enzyme variants like monoamine oxidase-N.
Implementation Method 1
amplification of the nucleic acid molecule using a population of mutagenic primers encoding the desired alternative (Boolean OR-type) mutations
Data Source
AI summary
This invention relates to a novel method for making a variant library, and to a variant library per se, preferably where the nucleic acid is DNA. The library is an OR-type library, wherein each variant of the library comprises an alternate (OR-type) mutation, using Boolean logic. These libraries, also referred to as OR-based or OR-type libraries or variant libraries comprising alternative mutations, are based upon the OR rule from Boolean logic to significantly reduce the overall size of the library, whilst still testing all the desired mutations.