Ligation-Assisted Sequencing of Long Nucleic Acids
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Solution Overview
Problem
Current nucleic acid sequencing methods are costly, time-consuming, and struggle with distinguishing between multiple DNA copies, particularly in applications like point-of-care pathogen detection and genetic analysis, where rapid, high-throughput, and cost-effective sequencing is needed, especially for long nucleic acid fragments and single nucleotide polymorphisms (SNPs).
Innovation Solution
A method involving controlled primer extension and hybridization, where sequencing primers are extended in limited cycles to generate incremental sequence reads, allowing for the assembly of the target nucleic acid sequence using labeled reversible terminators or ligation, enabling efficient sequencing of large genomes with low-cost equipment and high specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional sequencing methods are used, then sequencing can be performed, but the cost is high and the time consumption is long
Solution Approach 1:
The patent divides the sequencing process into multiple independent cycles, each cycle generating a portion of the sequence read. By segmenting the overall sequencing task into repeatable units (hybridization, extension, detection, release), the system can efficiently process long nucleic acid fragments without requiring excessive time or resources for each individual step.
Solution Approach 2:
The patent employs periodic cycling of hybridization, extension, and release steps to progressively build sequence reads. This periodic action allows the system to maintain high throughput by continuously cycling through the sequencing process with optimized timing for each step, reducing total time consumption while maintaining accuracy.
2Productivity
If conventional sequencing methods are used, then sequencing can be performed, but the cost is high
Solution Approach 1:
The patent designs the sequencing system to be self-sufficient by using the target nucleic acid itself as the template for primer extension and sequence determination. The method eliminates the need for expensive external reagents or complex equipment by leveraging the biological properties of the target molecule, thereby reducing material costs while maintaining high throughput.
Solution Approach 2:
The patent optimizes sequencing parameters such as primer concentration, extension time, and hybridization conditions to achieve high throughput with minimal resource consumption. By carefully controlling these parameters, the system maximizes the information obtained per unit of reagent and time, effectively reducing the cost per sequence read.
3Measurement precision
If standard sequencing methods are used, then sequencing can be performed, but it is difficult to distinguish between multiple DNA copies
Solution Approach 1:
The patent introduces sequencing primers as intermediaries that specifically bind to the target nucleic acid and serve as templates for sequence determination. These primers act as mediators between the detection system and the target DNA, enabling precise discrimination of sequence variations including single nucleotide polymorphisms by providing a defined starting point for extension that reflects the actual target sequence.
Solution Approach 2:
The patent applies local quality control by focusing the sequencing reaction on specific regions of the target nucleic acid through targeted primer binding. By concentrating the sequencing effort on particular loci of interest rather than attempting to sequence the entire genome uniformly, the method achieves high discrimination accuracy for specific variants while maintaining reliability across multiple DNA copies.
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 sequencing costs and increases speed, enabling the sequencing of large genomes and specific loci, including SNPs, with high accuracy and efficiency, suitable for point-of-care applications and field detection.
Implementation Method 1
hybridizing a second sequencing primer to the target nucleic acid
Implementation Method 2
extending a first sequencing primer hybridized to the target nucleic acid to generate a first primer extension product
Data Source
AI summary
The present invention provides methods and systems for sequencing long nucleic acid fragment. The present invention also provides a method of sequencing a target polynucleotide with fewer probes. Further, 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.