PINS Method for Rare DNA Enrichment and Sequencing
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Solution Overview
Problem
Current DNA-based methods face challenges in isolating and sequencing low-abundance target DNA fragments from mixed samples, leading to high costs and time consumption, especially in molecular diagnostics and metagenomics, where traditional methods require extensive sequencing and often rely on known sequences or culturable microorganisms.
Innovation Solution
The method involves serial dilution and replication of a mixed polynucleotide sample to increase the probability of detecting a target DNA molecule, followed by amplification and detection, allowing for the enrichment and sequencing of rare DNA fragments using techniques like Multiple Displacement Amplification (MDA) and PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing methods are used on mixed DNA samples, then complete sequencing coverage can be achieved, but the cost and time consumption increase significantly
Solution Approach 1:
The patent extracts and enriches specific target DNA sequences from complex mixed samples before sequencing. By using hybridization capture with biotinylated probes to selectively bind and isolate target sequences, the method removes unwanted background DNA, allowing focused sequencing of only the relevant portions, thereby reducing both time and cost while maintaining complete sequencing coverage of target regions
Solution Approach 2:
The patent performs preliminary enrichment and purification of target DNA sequences before the actual sequencing step. Through pre-amplification, hybridization capture, and selective purification steps, the target sequences are prepared in advance at high concentration and purity, which eliminates the need for extensive sequencing of background DNA and significantly reduces the overall sequencing time and cost
2Measurement precision
If traditional sequencing methods are used on mixed DNA samples, then all DNA sequences can be analyzed, but the cost increases significantly
Solution Approach 1:
The patent extracts and enriches specific target DNA sequences from complex mixed samples before sequencing. By using hybridization capture with biotinylated probes to selectively bind and isolate target sequences, the method removes unwanted background DNA, allowing focused sequencing of only the relevant portions, thereby reducing both time and cost while maintaining complete sequencing coverage of target regions
Solution Approach 2:
The patent performs preliminary enrichment and purification of target DNA sequences before the actual sequencing step. Through pre-amplification, hybridization capture, and selective purification steps, the target sequences are prepared in advance at high concentration and purity, which eliminates the need for extensive sequencing of background DNA and significantly reduces the overall sequencing time and cost
3Speed
If PCR-based methods are used to detect specific sequences, then rapid detection is achieved, but the method is limited to known sequences and requires precise integration site information
Solution Approach 1:
The patent employs a universal hybridization capture approach that can detect both known and unknown sequences. By using probes targeting conserved regions (such as antibiotic resistance genes) that are present across multiple species and variants, the method achieves rapid detection capability while simultaneously maintaining versatility to detect sequences without requiring precise integration site information, thus combining the advantages of both PCR speed and metagenomic broadness
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 significantly reduces the number of tests required to enrich target DNA, from hundreds of thousands to less than a few hundred samples, enabling efficient sequencing and diagnosis by increasing the frequency of the target DNA molecule, thus decreasing the sequencing burden and cost.
Implementation Method 1
serial dilution and replication of a mixed polynucleotide sample to increase the probability of detecting a target DNA molecule
Implementation Method 2
amplification and detection, allowing for the enrichment and sequencing of rare DNA fragments using techniques like Multiple Displacement Amplification (MDA)
Implementation Method 3
detecting the presence or absence of said target DNA molecule in said replicate dilution samples amplified in step (d)
Data Source
AI summary
The present invention pertains to an in vitro method in which the frequency of the targeted nucleotide sequence containing the DNA fragment of interest is increased stepwise, by several rounds of 1) dilution of a sample containing the DNA fragment of interest into several replicates (separation), 2) randomly amplifying DNA in the replicates (concentration), 3) detecting the DNA fragment of interest in at least one of the diluted and amplified replicates (selection) and repeating steps 1) through 3) until the DNA fragment of interest can be sequenced by standard sequencing techniques.