Reduced DNA Library Amplification for High-Yield Nucleic Acid Retrieval
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Solution Overview
Problem
Current methods for retrieving sequence-verified nucleic acid fragments after next-generation sequencing are inefficient, particularly due to high costs and low retrieval yields, especially when dealing with large DNA libraries where error-free fragments are difficult to selectively amplify amidst a vast population of error-containing fragments.
Innovation Solution
A method involving the reduction of DNA libraries before amplification, using sealing chambers to amplify sequence-verified nucleic acid fragments, and collecting single-stranded fragments separated during sequencing to create reduced libraries, which are then amplified and retrieved, thereby increasing the yield of desired nucleic acid fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to retrieve sequence-verified nucleic acid fragments after next-generation sequencing, then the retrieval process can be performed, but the retrieval yield is low and the cost is high when dealing with large DNA libraries
Solution Approach 1:
The patent applies preliminary action by performing library reduction before amplification. The method reduces the complex DNA library to a reduced library containing only sequence-verified fragments before the amplification step, thereby improving retrieval yield while reducing the burden on subsequent processing steps. This preliminary reduction step eliminates the need to amplify and then sort through vast numbers of error-containing fragments.
Solution Approach 2:
The patent segments the nucleic acid amplification process into distinct stages: library reduction, sealing chamber formation, and amplification. By separating the library reduction step from the amplification step and performing them in different conditions (with library reduction occurring before sealing), the method achieves better control over library complexity and improves overall retrieval efficiency.
2Measurement precision
If barcode sequences are used to selectively amplify desired nucleic acid fragments from large DNA libraries, then selective retrieval is possible, but the retrieval yield remains low due to the vast population of libraries
Solution Approach 1:
The patent performs library reduction before amplification, creating a reduced library that contains only sequence-verified fragments. This preliminary action eliminates the need to selectively amplify desired fragments from a vast population, as the reduction step has already enriched for the correct sequences. The subsequent amplification then operates on a much smaller, pre-enriched pool, dramatically improving retrieval yield while maintaining selectivity.
3Ease of manufacture
If chemically synthesized nucleic acid molecules are used for gene synthesis without further processing, then the process is simple, but error-free nucleic acid molecules cannot be distinguished from error-containing molecules
Solution Approach 1:
The patent performs library reduction as a preliminary step before amplification and gene synthesis. This reduction step uses sequence verification to identify and enrich error-free nucleic acid fragments before they are amplified and used for gene synthesis. By performing this verification and enrichment step preliminarily, the method maintains relative process simplicity while dramatically improving the reliability of the final synthesized genes.
Solution Approach 2:
The library reduction step enables the nucleic acid pool to self-select and enrich for error-free sequences through sequence verification. Rather than requiring external intervention to identify and separate error-free fragments, the verification process allows the correct sequences to be naturally enriched in the reduced library, which then serves as the template for subsequent amplification and synthesis.
Data Source
AI summary
A method for retrieving nucleic acid fragments, including: providing reduced DNA libraries during sequencing; amplifying nucleic acid fragments from the reduced DNA libraries; and retrieving the amplified nucleic acid fragments. The method further includes acquiring desired nucleic acid fragments among the retrieved nucleic acid fragments. The desired nucleic acid fragments are acquired using sequences having a homology to the barcode sequences, when the amplified nucleic acid fragments are tagged with specific barcode sequences.