Circular DNA Adaptor Insertion via Selective Methylation
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Solution Overview
Problem
Current sequencing methods face challenges in achieving low-cost, high-throughput sequencing and re-sequencing of genomic DNA, particularly in protecting restriction endonuclease recognition sites and ensuring accurate representation of sequences near these sites, while also allowing for the insertion of multiple DNA adaptors in defined positions.
Innovation Solution
The method involves repeated cycles of nucleic acid cleavage and ligation to insert multiple DNA adaptors into circular target DNAs at defined positions, using the same restriction endonuclease recognition site for all adaptors and protecting these sites to prevent excision and ensure accurate sequence representation, allowing for consecutive insertion of adaptors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If restriction endonuclease recognition sites are used in adaptors for consecutive insertion, then adaptor insertion efficiency is improved, but the sites in previously-inserted adaptors are inadvertently cleaved by the restriction enzyme
Solution Approach 1:
The patent applies preliminary anti-action by treating the recognition sites in previously-inserted adaptors with a methyltransferase enzyme before the restriction digestion step. This methylation modifies the sites in advance to prevent them from being recognized and cleaved by the restriction endonuclease, thereby protecting the integrity of previously-inserted adaptors while allowing new adaptors to be inserted at untreated sites.
Solution Approach 2:
The patent implements local quality by differentiating the treatment of recognition sites based on their location and state. Sites in previously-inserted adaptors are methylated and protected, while sites in target DNA and newly-added adaptors remain unmethylated and available for cleavage. This localized differentiation allows the same restriction enzyme to act selectively on different sites within the same sample.
2Reliability
If methylation is used to protect recognition sites in previously-inserted adaptors, then sequence accuracy is improved, but additional processing steps are required
Solution Approach 1:
The patent introduces a methyltransferase enzyme as an intermediary agent that temporarily modifies recognition sites in previously-inserted adaptors. This intermediary step adds a methyl group to protect the sites from restriction enzyme cleavage, and the modification can be removed or bypassed in subsequent steps. The intermediary approach enables selective protection without permanently altering the DNA sequence or requiring complex equipment.
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 enables efficient and accurate massively-parallel nucleic acid sequencing by protecting restriction endonuclease recognition sites and allowing for the consecutive insertion of adaptors, thereby improving the quality and quantity of sequencing data.
Implementation Method 1
digesting the library constructs with a restriction endonuclease that recognizes the restriction endonuclease recognition sites in the first adaptor
Implementation Method 2
methylating restriction endonuclease recognition sites in the library constructs
Data Source
AI summary
Aspects described and claimed herein provide methods to insert multiple DNA adaptors into a population of circular target DNAs at defined positions and orientations with respect to one another. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.