Amplifying Sheared DNA Fragments via Hairpin Adapters
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Solution Overview
Problem
Current methods for amplifying sheared DNA fragments from biofluid samples, such as those found in cancer diagnostics, are costly and require complex data analysis, with a need for more unbiased genome-wide views and reliable amplification of nuclease-sheared nucleic acids.
Innovation Solution
A method involving dephosphorylation, digestion with sequence-specific tools, ligation to hairpin looped adapters, removal of incompletely ligated products, and amplification using primers hybridizing to the adapters, which can be performed in a single buffer system and is applicable to degraded DNA, including ctDNA and methylated DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome sequencing of circulating free DNA is performed, then a comprehensive view of the tumor profile is achieved, but the cost and data analysis complexity increase significantly
Solution Approach 1:
The patent extracts and enriches only the mutation-bearing DNA sequences from the complex circulating free DNA sample using normalization techniques. This selective extraction approach allows comprehensive tumor profiling without requiring full whole genome sequencing, thereby reducing data analysis complexity while maintaining measurement precision for cancer detection.
2Reliability
If conventional amplification methods are used for sheared DNA fragments, then amplification is achieved, but the methods are costly and require complex procedures
Solution Approach 1:
The patent merges multiple steps including dephosphorylation, restriction enzyme digestion, adapter ligation, and amplification into a single integrated workflow that can be performed in one reaction vessel. This consolidation reduces procedure complexity while maintaining amplification reliability for sheared DNA fragments from biofluid samples.
Solution Approach 2:
The patent develops a universal amplification method that works for various types of degraded DNA including ctDNA, cfDNA, and methylated DNA using the same set of reagents and conditions. This multi-functional approach eliminates the need for separate protocols for different DNA types, reducing overall procedure complexity while ensuring reliable amplification across all sample types.
3Quantity of substance
If mutation-bearing sequences are enriched, then sequencing cost is reduced, but the enrichment process must be reliable for degraded DNA
Solution Approach 1:
The patent performs preliminary dephosphorylation and restriction enzyme digestion steps before adapter ligation and amplification. This preliminary processing of degraded DNA fragments ensures that the subsequent enrichment and sequencing processes work effectively on the prepared samples, maintaining enrichment reliability while reducing sequencing costs by pre-processing the complex DNA mixtures.
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 reliable amplification of sheared DNA fragments, reducing sample complexity and facilitating cost-effective sequencing by enriching mutation-bearing sequences, thereby enhancing the detection and prognosis of cancer.
Implementation Method 1
dephosphorylating the sheared DNA in the sample
Implementation Method 2
digesting the resulting DNA using at least one sequence specific DNA cutting tool
Implementation Method 3
ligating the digested nucleotide sequences to hairpin looped adapters
Implementation Method 4
removing incompletely ligated products
Implementation Method 5
amplifying the resulting nucleotide sequences using primers hybridizing to the adapters
Data Source
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AI summary
The present invention provides methods and kits for preparing a collection of degraded DNA fragments isolated from a biofluid sample of an individual. The prepared collection of nucleic acid sequences can be used in a diagnostic method such as for example detection and/or prognosis of cancer.