Template-Free Polynucleotide Synthesis with Failure Sequence Removal
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current template-free enzymatic methods for polynucleotide synthesis face challenges in separating full-length final products from failure sequences due to incomplete monomer coupling, deblocking, and capping, leading to the generation of truncated sequences.
Innovation Solution
The method involves hybridization-based and nuclease digestion steps to separate failure sequences by annealing primers to 3′-ends, extending with template-dependent polymerases, increasing reaction stringency to dissociate failure sequences, and using exonucleases to digest single-stranded DNA, along with capping and exonuclease-resistant modifications to protect full-length strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If template-free enzymatic synthesis is performed with repeated monomer coupling and deblocking cycles, then polynucleotide sequences can be formed, but failure sequences are generated due to incomplete reactions
Solution Approach 1:
A common primer binding site is added to the 3' end of each polynucleotide during synthesis. This preliminary action enables subsequent selective amplification and separation of full-length products from failure sequences through primer annealing and PCR, allowing the synthesis process to proceed efficiently while providing a mechanism for later purification.
Solution Approach 2:
The primer binding site is specifically positioned at the 3' end of polynucleotides, creating a localized feature that distinguishes full-length products from failure sequences. This local modification enables selective recognition and amplification of correctly synthesized sequences without affecting the overall synthesis efficiency.
2Manufacturing precision
If hybridization stringency is increased to dissociate failure sequences, then separation purity improves, but full-length product yield may be reduced
Solution Approach 1:
Full-length polynucleotides are amplified by annealing primers to the common primer binding sites and extending them to create reverse complements. This copying process generates double-stranded full-length products that can be selectively enriched through PCR, thereby increasing the yield of correct sequences without requiring excessively high hybridization stringency that would lose full-length products.
Solution Approach 2:
The common primer binding site acts as an intermediary element that facilitates selective identification and amplification of full-length polynucleotides. By providing a universal recognition sequence, it enables the use of PCR and other amplification techniques to enrich correct sequences without relying solely on hybridization stringency, thus balancing purity and yield.
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 significantly enhances the yield of full-length polynucleotides by effectively removing failure sequences, ensuring high purity and efficiency in the synthesis process.
Implementation Method 1
contacting under elongation conditions the initiator or elongated fragments having free 3'-O-hydroxyls with a 3'-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3'-O-blocked nucleoside triphosphate
Implementation Method 2
providing reaction conditions with a hybridization stringency that dissociate failure sequences among the double stranded polynucleotides
Implementation Method 3
providing reaction conditions with a hybridization stringency that dissociate failure sequences among the double stranded polynucleotides
Implementation Method 4
digesting strands of the dissociated double stranded polynucleotides
Data Source
AI summary
The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and/or nuclease digestion for removing failure sequences.