Circularizing Sequencing for Low-Frequency Mutation Detection
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Solution Overview
Problem
Current methods for high-throughput nucleic acid sequencing face challenges in accurately measuring low-frequency mutations due to high error rates, which hinder the precise detection and analysis of nucleic acid mutations, particularly in applications such as viral evolution, personalized medicine, and cancer research.
Innovation Solution
The method involves fragmenting isolated nucleic acids, denaturing them to form single-strand fragments, circularizing these fragments, and performing rolling circle amplification to produce high-yield amplification products with a sequencing resolution floor of at least 1×10−8 per base, using enzymes like micrococcal nuclease and RNA ligase, and avoiding DNA repair enzymes and high-temperature extraction processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional high-throughput sequencing methods are used, then sequencing speed and throughput are improved, but measurement precision of low-frequency mutations deteriorates due to high error rates
Solution Approach 1:
The patent applies preliminary action by performing multiple rounds of rolling circle amplification before sequencing to generate abundant circular DNA templates. This pre-amplification step ensures that even low-frequency mutations are sufficiently represented in the template pool, enabling accurate detection without requiring high sequencing throughput to compensate for low signal abundance
Solution Approach 2:
The patent uses rolling circle amplification to create multiple copies of circular DNA templates containing the original nucleic acid sequences. This copying process generates sufficient template material for high-precision mutation detection while maintaining the error profile of the original template, allowing accurate distinction between true mutations and sequencing errors
2Object-affected harmful factors
If DNA repair enzymes are used during amplification, then DNA damage is reduced, but measurement precision of true mutations deteriorates due to correction of authentic mutation signals
Solution Approach 1:
The patent explicitly extracts or removes DNA repair enzymes from the amplification system. By omitting these enzymes, the method preserves authentic mutation signals that would otherwise be corrected, while managing DNA damage through controlled amplification conditions and circularization protocols that minimize damage without requiring repair activity
Solution Approach 2:
The patent changes the parameter of enzyme selection by using polymerases without associated repair activities and optimizing reaction conditions to reduce DNA damage. This parameter change allows the system to tolerate some damage while preserving true mutations, achieving a balance that enables accurate mutation detection
3Productivity
If high-temperature extraction processes are used, then nucleic acid isolation efficiency is improved, but DNA damage increases leading to higher error rates
Solution Approach 1:
The patent changes the temperature parameter of the extraction process by using lower-temperature protocols. This parameter change reduces thermal damage to DNA while maintaining sufficient isolation efficiency through optimized chemical extraction conditions, preventing the introduction of artifacts that would interfere with mutation detection
4Speed
If rolling circle amplification is performed without circularization, then amplification speed is improved, but sequencing resolution deteriorates due to insufficient template uniformity
Solution Approach 1:
The patent performs circularization as a preliminary action before rolling circle amplification. This step creates uniform circular templates that serve as ideal substrates for rapid and uniform amplification, ensuring both high amplification speed and consistent template quality for high-resolution sequencing
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 the detection of mutation rates significantly lower than expected, with a high yield and resolution suitable for various biological applications, including cancer and microbial mutation analysis, while reducing sequencing errors and DNA damage.
Implementation Method 1
the fragmenting comprises contacting the isolated nucleic acids with a fragmentation enzyme (e.g., nucleases). In some embodiments, the fragmentation enzyme is fragmentase, which is a nickase paired to an endonuclease. In some embodiments, fragmentation enzyme is micrococcal nuclease (MNase).
Implementation Method 2
the denaturing comprises treating the nucleic acid fragments under conditions of alkaline pH. In some embodiments, the alkaline pH is greater than 11. In some embodiments, the alkaline pH is about 12.5.
Implementation Method 3
the circularizing comprises contacting the fragments with a ligase. In some embodiments, the ligase is an RNA ligase.
Implementation Method 4
amplifying circularized fragments with rolling circle amplification. In some embodiments, the methods produce at least about 1 microgram of amplification products.
Data Source
AI summary
The present disclosure provides methods related to nucleic acid amplification and sequencing. In particular, the present disclosure provides methods for amplifying and sequencing double stranded nucleic acids using a high yield and resolution circularizing-sequencing method.