Pre-Seeded Solid Supports for Monoclonal Nucleic Acid Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods face challenges due to insufficient quantity and quality of nucleic acids, leading to polyclonal populations that complicate data interpretation, especially in high-throughput and automated processes, and require inefficient amplification methods like RPA that often result in undesirable polyclonal populations.
Innovation Solution
A method involving pre-seeding and templating reactions using recombinase-polymerase amplification (RPA) to generate monoclonal nucleic acid populations on solid supports, where primers are attached to supports and amplified under isothermal conditions, followed by hybridization and extension to create templated supports for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If recombinase-polymerase amplification (RPA) is used to amplify nucleic acids, then amplification can be performed under isothermal conditions without thermal cycling, but the method generates undesirable polyclonal populations that complicate sequencing data interpretation
Solution Approach 1:
The amplification process is divided into two distinct phases: a pre-seeding phase that generates initial amplicons, and a templating phase that generates monoclonal populations from single-template amplicons. This segmentation allows the system to benefit from RPA's isothermal operation while achieving monoclonal purity through the sequential templating step.
Solution Approach 2:
The method performs preliminary amplification of template nucleic acid molecules before the actual templating reaction. By pre-seeding the supports with amplified templates, the system prepares monoclonal populations in advance, ensuring that subsequent sequencing reactions receive pure, single-template inputs that yield accurate results.
2Quantity of substance
If traditional amplification methods are used to increase nucleic acid quantity, then sufficient material can be obtained for sequencing, but the methods are time-consuming and require complex thermal cycling procedures
Solution Approach 1:
The method replaces traditional thermal cycling mechanical systems with isothermal RPA chemistry. By using recombinase and polymerase enzymes that operate at constant temperatures, the system eliminates the need for repeated heating and cooling cycles, significantly reducing amplification time while maintaining sufficient nucleic acid quantity for sequencing.
3Quantity of substance
If polyclonal nucleic acid populations are used in sequencing, then sufficient material can be provided, but the presence of multiple different templates complicates data interpretation and reduces sequencing accuracy
Solution Approach 1:
The method extracts and isolates single-template amplicons from the broader polyclonal population through the templating reaction. By selecting and amplifying only those amplicons that template from single original molecules, the system extracts pure monoclonal populations that maintain sequencing accuracy while providing sufficient material through the pre-seeding amplification step.
4Difficulty of detecting and measuring
If nucleic acid amplification is performed to enable sequencing of single templates, then sequencing sensitivity is improved, but the amplification process introduces polyclonal contamination that reduces manufacturing precision
Solution Approach 1:
The method introduces solid supports as intermediaries between the template nucleic acids and the amplification system. By attaching primers to these supports and performing templating reactions in situ, the system prevents cross-contamination between different templates while maintaining high sensitivity for single-template detection. The support acts as a physical barrier that isolates each amplification event.
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 produces high-quality, longer sequencing reads with reduced duplicates and errors, enhancing workflow efficiency and automation by confining and amplifying monoclonal nucleic acids, thereby improving sequencing results.
Implementation Method 1
extending the 3' end of the immobilized oligonucleotide primer portion of the partially double-stranded nucleic acids by template-dependent nucleic acid synthesis
Implementation Method 2
contacting single strands of the nucleic acid products of step (C) with supports having immobilized thereto a plurality of single-stranded oligonucleotide primers that are substantially identical to the fourth nucleotide sequence under annealing conditions thereby hybridizing single strands
Data Source
Figure 1A~1D
Figure 2A~2F
Figure 3A~3F
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.