Solid Support Capture Primers for Amplicon Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing techniques are laborious, time-consuming, and costly due to the need for extensive sample preparation and library construction, limiting throughput and increasing costs.
Innovation Solution
A method involving PCR amplification and direct immobilization of target polynucleotides onto a solid support using adapter-containing primers, followed by hybridization with universal and target-specific capture primers, allowing for clonal amplification and sequencing without the need for amplicon purification or additional enrichment steps, enabling rapid and efficient sequencing with minimal input nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If commercial sequencing techniques are used, then sequencing can be performed, but sample and library preparation are laborious and time-consuming
Solution Approach 1:
The patent applies preliminary action by pre-immobilizing capture probes onto solid supports before sample processing. This allows direct hybridization and enrichment of target nucleic acids without requiring laborious library preparation steps, significantly reducing sample preparation time while maintaining high sequencing throughput
Solution Approach 2:
The patent extracts and eliminates unnecessary intermediate steps from conventional sequencing workflows. By using directly immobilized capture probes, it removes the need for amplicon purification and additional enrichment steps, streamlining the process to achieve rapid sequencing with minimal input nucleic acid
2Measurement precision
If extensive sample preparation and library construction are performed, then sequencing quality can be maintained, but costs increase
Solution Approach 1:
The patent applies local quality by concentrating sequencing resources on specific target regions through capture probe hybridization. This localized enrichment approach maintains high sequencing quality for target regions while eliminating the need for comprehensive library construction, reducing overall process complexity and cost
Solution Approach 2:
The patent uses universal capture probes that can bind to multiple target sequences with common features. This multi-functional approach allows a single probe set to enrich for diverse target polynucleotides, maintaining sequencing quality across different targets while simplifying the preparation protocol
3Reliability
If conventional sequencing methods are used, then adequate coverage can be achieved, but input nucleic acid requirements are high
Solution Approach 1:
The patent uses immobilized capture probes as intermediaries to concentrate target nucleic acids from limited input samples. These probes selectively bind and enrich target sequences, enabling high detection sensitivity and adequate coverage even with very small amounts of input nucleic acid (10 ng or less)
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 achieves high-quality sequencing results with 85% or more uniformity, reducing sequencing time and cost by eliminating inefficient steps and allowing for the use of very small nucleic acid inputs, such as 10 ng or less, while improving detection sensitivity and accuracy.
Implementation Method 1
contacting a nucleic acid sample including a plurality of target polynucleotides with at least one primer under conditions sufficient for hybridization
Implementation Method 2
amplifying by polymerase chain reaction (PCR) the plurality of target polynucleotides to produce a plurality of amplicons
Implementation Method 3
directly contacting a plurality of target specific capture primers immobilized on a solid support with the plurality of amplicons under conditions sufficient for hybridization to produce a first plurality of immobilized amplicons
Implementation Method 4
extending the plurality of target specific capture primers to produce a plurality of immobilized extension products complementary to the target polynucleotides
Implementation Method 5
annealing the plurality of universal capture primers to the plurality of the immobilized extension products
Data Source
AI summary
The present disclosure relates to the field of molecular biology and more specifically to methods for capturing, amplifying and sequencing target polynucleotides on a solid surface.