Cell-Free DNA Library Construction via Direct Transposase Treatment
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Solution Overview
Problem
Current methods for non-invasive prenatal testing and early cancer detection using cell-free DNA from blood face challenges such as low DNA content leading to DNA loss during library construction, loss of nucleosome distribution information, and high sequencing costs.
Innovation Solution
A method involving direct treatment of body fluid samples with enzymes like transposase or endonuclease to fragment cell-free DNAs, adding adapters, and amplifying them to construct a library, which retains nucleosome DNA information and reduces sequencing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cell-free DNAs are extracted from body fluid samples before library construction, then the library construction process can be performed, but DNA loss occurs during extraction leading to reduced detection sensitivity
Solution Approach 1:
The patent eliminates the DNA extraction step by performing library construction directly on body fluid samples. The transposase enzyme acts on cell-free DNAs in the original sample matrix, extracting only the necessary enzymatic activity while leaving the DNA in its native state, thus avoiding extraction-induced DNA loss and improving detection sensitivity
Solution Approach 2:
The transposase enzyme serves as an intermediary that directly processes cell-free DNAs in the body fluid sample without requiring prior extraction. This mediator enables library construction while preserving the native DNA state, avoiding the harmful effects of extraction procedures
2Loss of information
If conventional library construction methods are used on extracted cell-free DNAs, then libraries can be constructed, but nucleosome distribution information is lost
Solution Approach 1:
The patent performs transposase treatment on the original body fluid sample before any extraction or processing steps. This preliminary action preserves the native nucleosome distribution state of cell-free DNAs, allowing subsequent library construction to retain epigenetic information including nucleosome positioning data
Solution Approach 2:
The patent changes the processing state of the sample from 'extracted DNA' to 'native body fluid sample with transposase treatment'. This parameter change maintains the physiological context and nucleosome distribution patterns, enabling preservation of epigenetic information while still allowing library construction
3Measurement precision
If methylation sequencing is performed on blood cell-free DNAs, then tissue traceability can be achieved, but complex steps cause DNA methylation information loss and low sequencing quality
Solution Approach 1:
The patent extracts only the transposase enzymatic activity from complex methylation sequencing protocols, performing a simplified treatment that preserves DNA methylation information. This selective extraction of the essential function reduces procedural complexity while maintaining measurement precision for tissue traceability
Solution Approach 2:
The patent applies transposase treatment specifically to cell-free DNAs in the body fluid sample, creating a localized modification that preserves global DNA methylation patterns. This local action maintains the quality of epigenetic information while simplifying the overall process compared to comprehensive methylation sequencing protocols
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 method enables efficient library construction from minute DNA amounts, retains nucleosome DNA information, and reduces sequencing costs, enhancing tissue traceability and epigenetic analysis for prenatal diagnosis and cancer detection.
Implementation Method 1
utilizing a transposase to treat the body fluid sample containing the cell-free DNAs, such that the cell-free DNAs are fragmented and added with an adapter through a transposition reaction
Implementation Method 2
utilizing an endonuclease to treat the body fluid sample containing the cell-free DNAs, such that the cell-free DNAs are fragmented by the endonuclease
Implementation Method 3
amplifying the fragmented DNAs obtained in step 1) to obtain the library of cell-free DNAs in the body fluid sample
Data Source
AI summary
A method for constructing a library of cell-free DNAs in body fluids, comprising directly acting a transposase or an endonuclease on a body fluid sample, fragmenting the cell-free DNAs within, and performing amplification to obtain a library. Also provided is a test kit using the present method for prenatal diagnosis or early detection of cancer.