Oligonucleotide Constructs with Unique Identifiers for Error-Free DNA Assembly
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Solution Overview
Problem
The construction of multi-kilobase scale DNA molecules is difficult and costly due to high error rates in de novo chemical synthesis and amplification steps, which complicates the selection of error-free oligonucleotides for synthetic biology applications.
Innovation Solution
The development of oligonucleotide constructs with unique identifiers and amplification/excision sites allows for the accurate selection and isolation of error-free regions of interest (ROIs), enabling high-throughput and high-accuracy sequence validation and assembly of larger DNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If de novo chemical synthesis is used to produce oligonucleotides, then large amounts of DNA building blocks can be produced cost-effectively, but the error rate increases significantly
Solution Approach 1:
The patent segments the DNA construction process into multiple steps: synthesizing many short oligonucleotides (20-200 nucleotides) in parallel, then assembling them into longer sequences. This allows high-throughput production of building blocks while managing error rates by keeping individual synthesis segments short and controllable.
Solution Approach 2:
The patent introduces unique identifiers as intermediary elements attached to each oligonucleotide. These identifiers enable tracking and validation of sequence accuracy through amplification and sequencing steps, allowing error detection without compromising the production of large quantities of DNA building blocks.
2Quantity of substance
If amplification steps are used to increase starting material, then sufficient DNA quantity is available for analysis, but errors are introduced during amplification
Solution Approach 1:
The patent implements feedback mechanisms where unique identifiers on amplified products are sequenced and compared to original designs. This feedback loop allows identification and rejection of amplification errors, ensuring that only accurate sequences proceed to final assembly, thus maintaining fidelity while enabling necessary amplification.
Solution Approach 2:
The patent replaces reliance on purely chemical synthesis with a hybrid approach incorporating biological validation steps (amplification and sequencing). This substitution allows detection and correction of errors that cannot be caught by chemical synthesis alone, improving overall reliability.
3Reliability
If traditional purification methods (gel purification, bacterial cloning) are used to identify error-free oligonucleotides, then sequence accuracy can be verified, but the process becomes complex and costly
Solution Approach 1:
The patent creates universal primer binding sites that work across all oligonucleotide variants, allowing a single amplification protocol to validate numerous sequences simultaneously. This multi-functional approach replaces multiple specialized purification steps with one unified validation process, reducing complexity while maintaining accuracy.
Solution Approach 2:
The patent uses amplification to create multiple copies of each unique oligonucleotide sequence, enabling statistical validation through sequencing. By copying sequences and analyzing consensus patterns across many copies, the method identifies error-free sequences more efficiently than traditional single-copy purification methods.
4Measurement precision
If comprehensive DNA sequencing is performed to identify error-free oligonucleotides, then sequence fidelity can be confirmed, but the cost and time requirements increase
Solution Approach 1:
The patent performs preliminary validation by attaching unique identifiers during the synthesis stage, before amplification and sequencing. This preliminary action allows rapid identification of candidate sequences that need full sequencing validation, reducing the overall number of sequences requiring time-consuming comprehensive analysis.
Solution Approach 2:
The patent employs a tiered validation approach where not all oligonucleotides undergo complete sequencing. Instead, unique identifiers enable selective validation of only those sequences that pass initial screening, performing partial validation on most sequences and excessive (full) validation only when necessary, thus optimizing time and cost.
Data Source
AI summary
The present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like.