Locus-Specific Sequencing with Capture Probes and Ligation
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Solution Overview
Problem
Current DNA sequencing methods face challenges in achieving high specificity and throughput for multiplex analysis, particularly in distinguishing between multiple DNA copies and detecting small amounts of target sequences, which is crucial for applications like paternity testing, forensic science, and genetic disease diagnosis.
Innovation Solution
A locus-specific sequencing method that involves forming hybridization complexes with capture probes and solution probes, followed by ligation and cap removal to enhance specificity, allowing for sequencing of targeted regions with high accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional DNA sequencing methods are used, then sequencing coverage can be achieved, but specificity is reduced and probe requirements increase
Solution Approach 1:
The sequencing process is divided into multiple targeted steps: hybridization with capture probes, ligation with adapter sequences, and selective amplification. This segmentation allows each step to be optimized for specificity, reducing the need for excessive probes while maintaining high sequencing accuracy.
Solution Approach 2:
Capture probes are designed with specific sequences that hybridize to target regions before sequencing begins. This preliminary hybridization step enriches the target DNA fragments, allowing subsequent steps to proceed with higher specificity and fewer probes required.
2Productivity
If multiplex analysis is performed to increase throughput, then analysis scale improves, but difficulty in detecting and measuring increases
Solution Approach 1:
The method employs universal adapter sequences that can be ligated to multiple different target regions. These universal adapters serve as common priming sites for subsequent amplification and sequencing, allowing multiple targets to be processed simultaneously through a unified workflow, thereby increasing throughput without proportionally increasing detection complexity.
Solution Approach 2:
Adapter sequences act as intermediaries between the capture probes and the sequencing primers. These adapters provide standardized binding sites that simplify the detection process across multiple targets, reducing the complexity that would otherwise arise from directly detecting each individual target region.
3Measurement precision
If hybridization conditions are optimized for specificity, then measurement precision improves, but hybridization efficiency decreases
Solution Approach 1:
The method employs capture probes of optimized length (typically 20-50 nucleotides) with specific GC content and melting temperature ranges. These parameter optimizations ensure that hybridization occurs with high specificity under defined conditions, while the subsequent ligation and amplification steps compensate for any reduction in hybridization efficiency.
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 method enables rapid, high-throughput sequencing with high specificity, capable of sequencing at least 50% of a genome using fewer probes than traditional methods, while avoiding minor allele frequencies and achieving high hybridization specificity.
Implementation Method 1
forming a hybridization complex, which comprises a first set of capture probes, a target polynucleotide, and a first solution probe. At least one of the first capture probes binds a first region of the target polynucleotide. The first solution probe binds a second region of the target polynucleotide.
Implementation Method 2
ligating at least one of the first set of capture probes and the first solution probe
Data Source
AI summary
The present invention provides methods for targeted sequencing of polynucleotide. In one aspect, the present invention provides a method of sequencing a target polynucleotide with fewer probes. In another aspect, the present invention provides a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing method are also provided in the present invention. The methods of the present invention allow low-cost, high-throughput and accurate sequencing of nucleic acids.