Circular Chimeric Nucleic Acid Capture for Low-Abundance Variant Detection
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Solution Overview
Problem
Current sequencing technologies struggle to accurately detect low-abundance mutations, methylation changes, and copy number variations in nucleic acids from blood samples with high specificity and sensitivity, particularly in the presence of a majority of normal nucleic acids, leading to high error rates and the inability to distinguish true mutations from PCR errors or false signals.
Innovation Solution
A method involving the creation of circular chimeric single-stranded nucleic acid constructs using linkers and oligonucleotide probes, followed by rolling circle amplification and sequencing, which allows for the targeted capture and highly sensitive detection of mutations, methylation changes, and copy number variations by utilizing hybridization, ligation, and polymerase extension to minimize false positives and maximize specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is used to amplify target nucleic acid regions, then the sensitivity of detection is improved, but the error rate increases due to introduction of rare errors that are indistinguishable from true mutations
Solution Approach 1:
The patent performs ligation of probes to target nucleic acids before amplification. This preliminary ligation step creates a stable complex where the probe is covalently attached to the target, allowing subsequent amplification to occur only on correctly bound probes. This prevents amplification of spurious signals and reduces the introduction of false mutations during PCR, thereby maintaining both sensitivity and reliability.
Solution Approach 2:
The patent introduces oligonucleotide probes as intermediaries between the target nucleic acid and the amplification process. These probes hybridize to the target and serve as primers for amplification. This intermediary step allows for selective amplification of only those regions that have correctly bound probes, filtering out non-specific amplification events and reducing error rates while maintaining detection sensitivity.
2Measurement precision
If total sequencing is performed on plasma DNA to detect low-abundance mutations, then the detection sensitivity is improved, but the cost and data management burden become prohibitive
Solution Approach 1:
The patent extracts and enriches for specific target regions of interest using hybridization-based capture with designed oligonucleotide probes. By selecting only the relevant genomic regions (e.g., cancer-related genes, prenatal diagnosis targets) and removing the vast majority of non-target DNA, the method reduces sequencing complexity and cost while concentrating sequencing depth on the regions most likely to contain clinically relevant mutations.
Solution Approach 2:
The patent divides the genome into specific target regions of interest and uses separate probes for each region. This segmentation allows for focused sequencing of only the relevant portions of the genome, rather than attempting to sequence the entire genome. The probes are designed to cover specific genes or regions, enabling targeted enrichment that reduces data management burden and sequencing costs while maintaining high detection sensitivity for mutations in those regions.
3Productivity
If sequence capture is used to enrich target regions, then the sequencing cost is reduced, but dropout occurs where 90-95% of desired sequences are captured but some fragments are missing
Solution Approach 1:
The patent combines hybridization-based probe binding with covalent ligation to create a more robust enrichment method. The probe first hybridizes to the target sequence, then a ligation step covalently joins the probe to the target. This merging of two mechanisms (hybridization plus ligation) ensures more complete capture of target fragments compared to hybridization alone, reducing dropout while maintaining sequencing efficiency.
4Measurement precision
If bisulfite treatment is used to determine promoter methylation, then the methylation detection capability is improved, but the DNA sample is destroyed and the ability to identify multiple methylation changes is lost
Solution Approach 1:
The patent changes the chemical approach to methylation detection by using enzymatic digestion with methyl-sensitive restriction enzymes instead of bisulfite treatment. This parameter change allows for methylation detection without destroying the DNA sample. The enzymes specifically recognize and cut unmethylated DNA at certain sequences, allowing methylation status to be determined while preserving the DNA for additional analyses.
Solution Approach 2:
The patent enables the DNA sample to serve multiple functions by preserving its integrity after methylation analysis. The same DNA sample can be used for methylation detection, mutation analysis, and other genomic studies. This multi-functionality is achieved by using methods that do not destroy the DNA, allowing a single sample to provide comprehensive diagnostic information.
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
The method enables highly specific and sensitive detection of genetic alterations in blood samples, suitable for cancer screening and prenatal diagnosis, with reduced error rates and improved accuracy through tandem sequencing and rolling circle amplification, enabling early detection and monitoring of diseases.
Implementation Method 1
contacting a first oligonucleotide probe to the linker appended target nucleic acid, wherein said first oligonucleotide probe comprises: a portion complementary to a 3' linker portion of the linker appended target nucleic acid segment; and a portion complementary to the 5' linker portion of the linker appended target nucleic acid DNA segment wherein said contacting is under conditions effective for the 3' and 5' portions of the first oligonucleotide probe to hybridise to the complementary portions of the linker appended target nucleic acid segment
Implementation Method 2
amplifying the collection of circular chimeric single stranded nucleic acid constructs by rolling circle amplification primed from the second primer to form a primary extension product
Implementation Method 3
amplifying the collection of circular chimeric single stranded nucleic acid constructs by rolling circle amplification primed from the second primer to form a primary extension product
Data Source
Figure 1A~1D
Figure 2A~2C
Figure 3A~3C
AI summary
The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, IncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, IncRNA's splice-site variants, translocations, and miRNA.