OREO-seq eccDNA Amplification via OriC Transposome Tagging
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Solution Overview
Problem
Current methods are inadequate for efficiently amplifying and characterizing human extrachromosomal circular DNA (eccDNA), which is crucial for understanding its biological functions and potential role in diseases such as cancer.
Innovation Solution
The OREO-seq method uses an in vitro assembled transposome carrying a replication origin sequence from E. coli (oriC) to specifically tag and exponentially amplify eccDNAs from eukaryotic cell extracts. This method involves engineering a transposon with oriC and transposases, introducing it to cellular genetic material, and exploiting the isothermal replication cycle reaction to selectively amplify eccDNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional amplification methods are used on eccDNA, then chromosomal DNA is co-amplified along with eccDNA, but this reduces the specificity and resolution of eccDNA sequencing
Solution Approach 1:
The method segments the amplification process by introducing an oriC tag that specifically marks eccDNA molecules, allowing them to be selectively amplified through isothermal replication while chromosomal DNA remains unamplified. This segmentation resolves the contradiction by separating eccDNA amplification from chromosomal DNA amplification.
Solution Approach 2:
The oriC sequence acts as an intermediary element that bridges eccDNA and the amplification system. By tagging eccDNA with oriC, the method enables specific recognition and amplification of eccDNA without affecting chromosomal DNA, thereby improving sequencing resolution without excessive complexity.
2Productivity
If transposome is introduced to amplify eccDNA, then eccDNA amplification efficiency increases, but the risk of amplifying linear chromosomal DNA products also increases
Solution Approach 1:
The method applies local quality by making the amplification capability specific to circular DNA structures through the oriC tag. The isothermal replication system is designed to recognize and amplify only circularized DNA with oriC, giving different amplification properties to circular vs linear DNA, thus improving reliability while maintaining productivity.
Solution Approach 2:
The method converts the potential harm of non-specific amplification into a benefit by using the circular structure of eccDNA as a selective advantage. The isothermal replication system preferentially amplifies circular DNA containing oriC while leaving linear chromosomal DNA untouched, turning the structural difference into a selective amplification mechanism.
3Measurement precision
If Exo V nuclease treatment is applied to remove linear products, then eccDNA resolution improves, but the processing time and complexity increase
Solution Approach 1:
The method performs preliminary action by designing the transposome to preferentially generate circular DNA products during the amplification process itself. This preliminary circularization reduces the amount of linear DNA requiring Exo V treatment, thereby improving resolution while minimizing the time loss associated with nuclease processing.
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
OREO-seq enables the selective amplification of eccDNAs over chromosomal DNA, allowing for higher resolution and efficient sequencing of these circular DNA species. This approach facilitates the characterization of eccDNAs and their potential role in disease, including cancer.
Implementation Method 1
exposing the genetic material to the conditions necessary for an isothermal replication cycle reaction causes the eccDNAs that possess the transposed OriC region to replicate while the chromosomal DNA does not
Implementation Method 2
it randomly binds and catalyzes a transposition reaction-inserting oriC into both chromosomal DNA as well as eccDNA
Implementation Method 3
treatment with an Exo V nuclease can be used to assist the breakdown of remaining linear products for higher resolution of amplified eccDNA or ecDNA
Implementation Method 4
the amplification products can be treated with PacI to digest circular background mitochondrial DNA
Data Source
AI summary
This invention provides compositions, reagents, methods, and kits for isolating, amplifying, and sequencing extrachromosomal circular DNA (eccDNA or ecDNA), particularly eukaryotic eccDNA and ecDNA. The various embodiments of the invention provide methods for exponential amplification of eccDNA or ecDNA and subsequent nucleic acid sequencing to enable speedy diagnosis of a sample from a patient and genomic screening for disease states therein and identifying and providing treatment thereof.