Two-Direction Nucleic Acid Probes for Mutant Sequence Capture
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Solution Overview
Problem
Existing nucleic acid enrichment methods, such as PCR and hybridization, suffer from amplification bias, inability to capture complementary strands, and inefficiencies in capturing mutant sequences, leading to deviations in mutation frequency detection.
Innovation Solution
Design of two-direction probes with sense and antisense strands of equal length without overlap, biotin labels for magnetic capture, and use of base-free spacer groups to capture both wild type and mutant sequences, ensuring minimal interaction and improved capture specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If one-direction probes are used for nucleic acid capture, then the probe design is simpler, but the complementary strand nucleic acid libraries cannot be captured resulting in loss of capture copy number
Solution Approach 1:
The probe set is segmented into two distinct directions: sense strand probes and antisense strand probes. Each direction targets complementary strands of the nucleic acid library, ensuring both strands are captured equally. This segmentation resolves the contradiction by dividing the capture function across two probe types rather than relying on a single probe design.
Solution Approach 2:
The probe design parameters are changed from single-direction to two-direction coverage. By adjusting the probe orientation parameter and implementing equal-length sense and antisense probes, the system achieves balanced capture of both complementary strands, doubling the effective capture copy number while maintaining manageable design complexity through standardized probe construction.
2Ease of manufacture
If probes are designed according to normal genome sequences, then the probe design is straightforward, but the normal genome sequences rather than the mutant sequences are preferentially captured resulting in deviation of mutation frequency
Solution Approach 1:
The probe design incorporates local quality adjustments by creating specific probe variants that target mutant sequences at particular locations. While the overall probe set is based on the normal genome sequence for ease of design, local modifications are made to include probes that specifically recognize and capture mutant alleles, ensuring accurate mutation frequency measurement without compromising the simplicity of the overall design approach.
Solution Approach 2:
Instead of designing probes only for the normal reference sequence, the approach inverts the strategy by also designing probes that specifically target mutant sequences. This allows the capture system to preferentially enrich mutant alleles alongside normal sequences, correcting the bias and enabling accurate mutation frequency detection while maintaining the simplicity of reference-based probe design.
3Productivity
If the amount of probes is increased to promote hybridization reaction, then the hybridization efficiency is improved, but the amount of sample required is reduced which may lead to loss of rare mutant sequences
Solution Approach 1:
The probe mixture is segmented into two equal components: sense strand probes and antisense strand probes. This segmentation ensures that both complementary strands of the nucleic acid library are captured with equal efficiency. By distributing the total probe amount across both directions rather than overwhelming the system with excess probes, the method maintains high hybridization efficiency while preserving rare mutant sequences that would be lost in sample dilution.
Solution Approach 2:
The probe design parameters are optimized to use equal-length sense and antisense probes, which changes the hybridization dynamics to favor balanced capture of both strands. This parameter adjustment improves overall hybridization efficiency without requiring excessive probe amounts, thereby preventing the dilution and loss of rare mutant sequences that would occur with large sample reductions.
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 two-direction probes enhance capture specificity and yield, increasing the number of original copies of nucleic acid captured, particularly mutant sequences, and improve mutation detection rates.
Implementation Method 1
The 3' or 5' of the probes have a biotin label that can bind the avidin on magnetic beads
Implementation Method 2
the target genes were captured by hybridization between probes and genomes
Data Source
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AI summary
Disclosed are a probe for nucleic acid enrichment and capture and a design method thereof. The probe consists of a sense strand probe and an antisense strand probe, wherein both the sense strand probe and the antisense strand probe are probes without an overlapping design, and both are 30-89 bases in length. The 3' or 5' of the probe is biotin-labeled and can bind to the avidin on the magnetic beads. The two-way probe can increase the specificity of the capture (i.e. reducing the capture of genomic DNA at non-target sites), can significantly increase the capture of the sample mutant DNA and can increase the original copy number of the captured sample nucleic acid.