Pre-Seeded Solid Supports for Monoclonal Nucleic Acid Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods face challenges in generating monoclonal populations for high-throughput analysis due to polyclonal contamination and insufficient sensitivity, leading to complex data interpretation and high costs.
Innovation Solution
A method involving pre-seeding and templating reactions using recombinase-polymerase amplification (RPA) to generate templated solid supports with substantially monoclonal nucleic acid molecules, followed by isothermal amplification to produce multiple copies of template nucleic acids, ensuring high-quality sequencing results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional nucleic acid amplification methods are used, then amplification can be achieved, but polyclonal contamination occurs and monoclonal purity is reduced
Solution Approach 1:
The amplification process is divided into multiple discrete steps: initial amplification to generate sufficient material, followed by selective capture of monoclonal populations through specific binding interactions, and subsequent expansion of only the desired clones. This segmentation allows separation of quantity generation from purity selection.
Solution Approach 2:
A capture reagent with specific binding affinity acts as an intermediary between the polyclonal amplification products and the final monoclonal population. This reagent selectively binds to and isolates the desired monoclonal nucleic acids from the polyclonal mixture, enabling purity separation without losing the amplified material.
2Productivity
If sequencing is performed on polyclonal populations, then sequencing can be conducted, but data interpretation becomes complex and sensitivity is reduced
Solution Approach 1:
Monoclonal purification is performed as a preliminary step before sequencing. By pre-isolating the monoclonal population from the polyclonal mixture through selective capture, the sequencing step receives purified input material, eliminating the need for complex data interpretation and improving detection sensitivity.
3Productivity
If high-throughput sequencing is implemented, then analysis efficiency increases, but cost increases
Solution Approach 1:
The method creates multiple copies of the target nucleic acid through selective amplification and capture cycles. By generating sufficient monoclonal copies through the capture and expansion process, the method achieves high-throughput sequencing capability while maintaining cost-effectiveness through efficient use of reagents and optimized reaction conditions.
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 enhances high-throughput sequencing by increasing the number of high-quality reads, reducing duplicate reads, and improving sequencing efficiency and accuracy while maintaining cost-effectiveness.
Implementation Method 1
utilizes enzymes to bind oligonucleotide primers to their complementary partners in duplex DNA
Implementation Method 2
recombinase-polymerase amplification (RPA), which is a DNA amplification process that utilizes enzymes to bind oligonucleotide primers to their complementary partners
Data Source
AI summary
The present disclosure provides methods, compositions and kits as well as systems for manipulating nucleic acids, including implementing isothermal amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using a pre-seeded solid support. Provided are rapid and efficient methods for generating template nucleic acid molecules comprising specific nucleotide sequence bound to solid support. Such methods can be used, for example, in manipulating nucleic acids in preparation for analysis methods that utilize monoclonal populations of nucleic acids.