Polynucleotide Variant Synthesis via Segmented Amplicon Assembly
Solution Overview
Problem
Current methods for generating polynucleotide variants with defined combinations of mutations are inefficient and costly, requiring whole gene synthesis and large numbers of oligonucleotide primers, which limits the ability to screen a large number of novel combinations due to lower yields and higher costs.
Innovation Solution
A method involving the separate amplification of a reference polynucleotide template with pairs of forward and reverse primers to generate an addressable library of amplicons, which are then assembled and replicated to produce polynucleotide variants with defined nucleotide differences, allowing for the synthesis of multiple variants with improved properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If whole gene synthesis and large numbers of oligonucleotide primers are used to generate polynucleotide variants, then the variants can be produced, but the cost increases and the number of variants that can be screened is limited
Solution Approach 1:
The patent divides the reference polynucleotide into multiple overlapping segments or fragments. Each segment is amplified separately using specific primer pairs, and then the segments are assembled through recombination to form complete polynucleotide variants. This segmentation allows systematic generation of multiple variants with defined mutation combinations without requiring synthesis of entire genes from scratch.
Solution Approach 2:
The patent performs preliminary amplification of segmented fragments from the reference polynucleotide using pairs of primers that introduce defined nucleotide differences. These pre-amplified segments are then ready for assembly through recombination. This preliminary action of creating a library of segmented fragments with embedded mutations enables efficient subsequent assembly of multiple variant combinations without repeated full gene synthesis.
2Manufacturing precision
If de novo synthesis techniques are used to generate polynucleotide variants, then variants with defined mutations can be produced, but the yield of correct sequences is lower
Solution Approach 1:
The patent uses recombination-based assembly where pre-amplified segments with defined mutations self-assemble into complete polynucleotide variants through homologous recombination. The system utilizes the natural recombination capability of the molecular assembly process to correctly join segments with defined nucleotide differences, reducing errors compared to traditional de novo synthesis and improving the yield of correct sequence variants.
Data Source
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AI summary
The present disclosure relates to methods for generating libraries of polynucleotide variants comprising defined nucleotide differences relative to a reference polynucleotide.