Loop Adapter Protection for Targeted DNA Enrichment
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Solution Overview
Problem
Current nucleic acid enrichment methods, such as PCR and probe capture, suffer from biases, inefficiencies, and high costs, particularly when targeting specific genes, and are limited by the length of sequencing reads and the need for extensive computational resources.
Innovation Solution
The use of loop adapters that protect target nucleic acids from exonuclease digestion, comprising sticky ends, hybridization segments, and a loop segment, allowing for efficient enrichment and sequencing of desired nucleic acid sequences using exonuclease protection and CRISPR/Cas systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR method is used for targeted DNA enrichment, then amplification of specific regions is achieved, but biases and errors are introduced that negatively influence downstream sequencing
Solution Approach 1:
The invention extracts only the necessary enrichment function from PCR by using exonuclease digestion to remove non-target DNA, while eliminating the PCR amplification step that introduces biases and errors. This allows selective enrichment without the harmful side effects of PCR.
Solution Approach 2:
The patent introduces loop adapters as intermediary molecules that hybridize to target DNA and protect it from exonuclease digestion. These adapters serve as mediators between the target DNA and the exonuclease enzyme, enabling selective protection of target sequences without requiring PCR amplification.
2Ease of manufacture
If probe capture method is used for targeted DNA enrichment, then sequencing library preparation is facilitated, but capture efficiency and specificity are low and it is limited to short DNA fragments
Solution Approach 1:
The invention changes the key parameter of DNA fragment length compatibility by using exonuclease digestion instead of probe hybridization. This allows the method to work with long DNA fragments that cannot be effectively captured by traditional probe methods, while maintaining ease of library preparation.
Solution Approach 2:
The patent replaces the mechanical probe hybridization system with an enzymatic exonuclease digestion system. This substitution enables higher capture efficiency and specificity by using enzyme-based recognition and digestion mechanisms rather than relying on probe-DNA hybridization stability.
3Loss of information
If whole genome sequencing is performed to search for variants in a few genes, then comprehensive genomic information is obtained, but sequencing resources and computational power are wasted
Solution Approach 1:
The invention extracts only the relevant genomic information by enriching for specific target regions using loop adapters and exonuclease digestion. This eliminates the need to sequence the entire genome when only a few genes are of interest, thereby reducing resource waste while maintaining information completeness for the targets of interest.
Solution Approach 2:
The patent applies partial action by performing enrichment only on the specific genomic regions of interest rather than sequencing the entire genome. This partial approach is sufficient for detecting variants in target genes while avoiding the excessive resource consumption of whole genome sequencing.
4Measurement precision
If Sanger sequencing method is used for genome sequencing, then accurate sequencing is achieved, but cost is high and throughput is low
Solution Approach 1:
The invention segments the genome into specific target regions using loop adapters that bind to predetermined locations. This segmentation allows parallel sequencing of multiple samples and regions using NGS technology, thereby increasing throughput while maintaining accuracy through targeted enrichment.
Solution Approach 2:
The patent creates a universal enrichment platform using loop adapters and exonuclease digestion that can be applied to any target region of interest. This multi-functional approach enables high-throughput sequencing of multiple different targets using the same methodology, unlike Sanger sequencing which is limited to single-region analysis.
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
Enriches target nucleic acids with high specificity and efficiency, reducing sequencing costs and computational demands, while maintaining nucleic acid length, and enabling long-read sequencing technologies.
Implementation Method 1
a nucleic acid loop adapter that can protect the target nucleic acid from exonuclease digestion. The loop adapters typically include (i) a sticky end nucleic acid segment designed to hybridize with a sticky end of a target linear nucleic acid
Implementation Method 2
treating the nucleic acid sample with one or more exonucleases, wherein loop adapter ligated nucleic acids are undigested by the exonuclease(s) and non-adapter ligated nucleic acids are digested by the exonuclease(s)
Data Source
AI summary
Compositions and methods for enriching DNA from any loci of interest are provided. Endonuclease(s) such as RNA-guided nucleases Cpf1/Cas9 can cut the flanking regions of the gene of interest in, for example genomic DNA, followed by ligation of loop adapters. The loop adapters protect the gene of interest for subsequent digestion with one or more exonucleases. Multiple genes of interest can be digested and ligated with loop adapters at the same time. The undigested target sequence can be further purified by, for example, gel extraction using, for example, a commercial DNA purification kit. The enriched DNA can be used for sequencing.