Layered Capture Primer Arrays for Targeted Monoclonal Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current next-generation sequencing methods lack the ability to perform targeted sequencing of specific polynucleotides, such as partial genomes, due to indiscriminate capture by universal capture primers, which is costly and inefficient for data handling.
Innovation Solution
A microarray design with distinct layers of capture primers, including first and second capture primer pairs, where the second pair is 3'-phosphate-terminated, allowing for targeted hybridization and amplification of specific polynucleotides, forming monoclonal populations of amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If universal capture primers are used for indiscriminate capture of genomic fragments, then whole genome sequencing can be performed, but targeted sequencing of specific polynucleotides is not enabled
Solution Approach 1:
The capture primer is divided into two distinct layers: a first layer containing universal capture primers for initial binding, and a second layer containing target-specific capture primers for selective amplification. This segmentation allows the system to perform both whole genome sequencing and targeted sequencing by controlling which layer is activated
Solution Approach 2:
The invention adds a vertical dimension to the capture primer structure by implementing a two-layer configuration on the solid surface. The first layer serves as a foundation for universal capture, while the second layer provides target-specific functionality, enabling selective sequencing without complicating the horizontal layout
2Quantity of substance
If indiscriminate capture of all genomic fragments is performed, then complete genome coverage is achieved, but data handling costs and complexity increase
Solution Approach 1:
The invention extracts and isolates only the desired target polynucleotides through the second layer of target-specific capture primers, separating them from the rest of the genome. This extraction approach reduces the quantity of data that needs to be handled while maintaining complete information about the target regions
Solution Approach 2:
The second layer of capture primers provides local quality control by being specific to particular target regions. This allows different regions of the genome to be captured with different specificities, enabling researchers to focus on regions of interest while reducing overall data complexity
3Reliability
If 3'-phosphate-terminated primers are used in the second layer, then targeted amplification is enabled, but primer functionality is restricted
Solution Approach 1:
The system dynamically controls primer functionality through the 3'-phosphate termination. The second layer primers are initially inactive (phosphate-terminated) to prevent premature amplification, but can be activated when needed. This dynamic control ensures that only target-specific priming occurs after the first layer has established initial binding
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
Enables targeted next-generation sequencing of partial genomes by creating monoclonal populations of amplicons, improving sequencing efficiency and reducing data handling costs.
Implementation Method 1
contacting a sample including a plurality of target polynucleotides with the substrate under conditions sufficient for a target polynucleotide to hybridize with a capture primer
Data Source
AI summary
The present disclosure relates to the field of molecular biology and more specifically to microarrays and methods. In particular, the present disclosure relates to a method for amplifying a nucleic acid on a substrate comprising at least one well, the method includes: depositing a plurality of first capture primer pairs and a plurality of second capture primer pairs in a layer at least partially covering the inner well surface, wherein the primer density of first capture primer pairs is higher than the primer density of second primer pairs; contacting a sample comprising a plurality of target polynucleotides with the substrate under conditions sufficient for a single target polynucleotide per well to hybridize with a capture primer of the second capture primer pair; and performing a kinetic exclusion assay (KEA) to produce a monoclonal population of amplicons from the single target polynucleotide.