Nucleic Acid Tagging for Mutant Detection Sensitivity
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Solution Overview
Problem
Current genetic analysis methods face challenges due to the short length of cell-free DNA, high error rates in next-generation sequencing, and low sensitivity in detecting mutant nucleic acid sequences amidst a vast number of wild-type sequences, particularly in biological samples.
Innovation Solution
A method involving the attachment of a nucleic acid 'tag' to a target sequence using single-stranded oligonucleotides, where hybridization brings the tag into proximity for covalent bonding, facilitating amplification, detection, and analysis by incorporating heteropolynucleotides and homopolynucleotides through terminal transferase activity and ligase action.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next generation sequencing is used for nucleic acid analysis, then sequencing capacity and throughput are improved, but error rate increases to 0.3% which reduces sensitivity and specificity
Solution Approach 1:
The patent applies preliminary action by performing targeted enrichment of mutant nucleic acid sequences before sequencing. The method uses probes to capture and concentrate rare mutant sequences from complex biological samples, ensuring that the sequences of interest are pre-selected and enriched prior to the sequencing step. This preliminary enrichment reduces the impact of sequencing errors on detection sensitivity.
Solution Approach 2:
The patent introduces an intermediary enrichment step using hybridization probes as mediators between the complex biological sample and the sequencing process. These probes specifically bind to mutant sequences, acting as intermediaries that selectively isolate target sequences from the vast background of wild-type sequences, thereby improving both sensitivity and reducing the relative impact of sequencing errors.
2Measurement precision
If hybridization-based enrichment is used to detect mutant sequences, then sensitivity is improved to 1 mutant per 2500 wild type molecules, but manufacturing complexity increases and practical sensitivity remains insufficient
Solution Approach 1:
The patent merges multiple functions into a unified enrichment and detection system. The method combines hybridization-based capture with streamlined processing steps, integrating sample preparation, enrichment, and detection into a more cohesive workflow. This merging reduces the number of separate manual operations and simplifies the overall process while maintaining high sensitivity.
Solution Approach 2:
The patent optimizes parameters such as probe design, hybridization conditions, and sequence length to improve sensitivity while reducing complexity. By carefully tuning these parameters, the method achieves practical sensitivity that overcomes the limitations of conventional hybridization approaches without requiring excessively complex procedures.
3Loss of information
If cell-free DNA is used as target, then biomarker information is improved, but DNA length decreases to median 168 bp which greatly affects sensitivity
Solution Approach 1:
The patent applies preliminary action by performing size-selected enrichment of cell-free DNA fragments before analysis. The method selectively captures and concentrates cfDNA fragments of specific size ranges, ensuring that the rare mutant sequences are pre-enriched and properly sized for optimal detection. This preliminary size-based selection improves sensitivity despite the inherently short length of cell-free DNA.
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
Enhances the detection and analysis of target sequences by improving sensitivity and specificity, allowing for accurate identification and measurement of mutant sequences amidst a high background of wild-type sequences.
Implementation Method 1
Hybridization of the target binding sequence to the target sequence brings the detectable sequence tag into proximity of the target sequence for formation of a covalent bond
Implementation Method 2
contacting a single stranded nucleic acid with a terminal transferase activity and a mixture of two or more different nucleotides under conditions in which nucleotides in the mixture are sequentially and randomly added to the 3' terminus of the nucleic acid
Implementation Method 3
contacting the second modified nucleic acid with a second single-stranded nucleic acid comprising a binding polynucleotide complementary to, or substantially complementary to, the homopolynucleotide in the second modified nucleic acid and a ligase activity under conditions in which the 3' end of the second modified nucleic acid and the 5' end of the second single-stranded nucleic acid ligate
Data Source
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AI summary
Provided in certain aspects are methods for dynamically adding molecular indexes to nucleic acid, and optionally analyzing the tagged nucleic acid. Also provided in certain aspects are methods for producing a single-stranded nucleic acid molecule from two molecules. The first molecule typically is a single-stranded nucleic acid (ssNA) containing a target sequence with optional linked nucleic acid sequences. The second molecule typically is a ssNA containing a target binding sequence and a nucleic acid sequence "tag" that is not complementary to the target sequence. The first and second ssNA molecules can be hybridized or annealed under conditions in which the target sequence and target binding sequence hybridize or anneal to each other by base pair complementarity, followed by contact of the hybridized or annealed molecules with a single-stranded nucleic acid ligase activity that ligates the 3'-end of the first ssNA molecule with the 5'phosphate (optionally adenylated) moiety of the second ssNA molecule to produce one product, a ligated ssNA molecule.