Surface-Bound Oligonucleotides for Target DNA Capture
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Solution Overview
Problem
Current sequencing methods, particularly re-sequencing, are labor-intensive and prone to biases due to complex molecular biology protocols, leading to increased sequencing errors and requiring large sample quantities.
Innovation Solution
A method involving the use of a substrate with randomly interspersed surface-bound oligonucleotides for capturing and amplifying specific genomic sequences, utilizing selection oligonucleotides to hybridize and extend primers, and employing bridge PCR for amplification, which simplifies sample preparation and reduces manipulation steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hybridization-based assays or in-solution techniques are used to capture target DNA, then target capture can be achieved, but complicated molecular biology protocols are required involving multiple enzymatic reactions, purification steps and gel electrophoresis
Solution Approach 1:
The patent combines target capture and sequencing library preparation into a single integrated process. Surface-bound oligonucleotides serve dual functions as both capture probes and sequencing primers, eliminating the need for separate purification and library preparation steps. The captured DNA fragments are directly amplified and prepared for sequencing in one continuous workflow on the solid support.
Solution Approach 2:
The patent extracts and eliminates unnecessary intermediate steps from traditional workflows. By performing amplification and library preparation directly on the solid support without eluting captured DNA, the method removes multiple purification steps, enzymatic reactions, and gel electrophoresis steps that are typically required in conventional target capture sequencing.
2Reliability
If multiple purification steps and enzymatic reactions are employed, then DNA enrichment can be achieved, but labor intensive sample preparation is required
Solution Approach 1:
The patent implements continuous amplification and library preparation directly on the solid support without interrupting the workflow. DNA fragments captured on surface-bound oligonucleotides undergo in-situ amplification and adapter ligation in a continuous process, eliminating repeated handling, purification, and transfer steps that consume time and labor.
3Reliability
If conventional target capture methods are used, then enriched DNA sample can be obtained, but sample manipulations cause bias in DNA content and increase sequencing error rate
Solution Approach 1:
The patent enables self-service amplification and library preparation directly on the solid support. Captured DNA fragments undergo in-situ amplification and adapter ligation without being transferred to solution, eliminating manipulation-induced biases and reducing contamination risks. The solid support environment maintains DNA integrity throughout the process.
4Productivity
If traditional sequencing library preparation is performed, then sequencing can be conducted, but large sample quantities are required due to loss during multiple manipulation steps
Solution Approach 1:
The patent performs preliminary amplification and library preparation steps directly on the solid support before sequencing. By establishing sufficient DNA copy numbers and completing library construction in-situ, the method ensures adequate material for sequencing without requiring large starting DNA quantities, as losses during transfer and purification are eliminated.
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 reduces sequencing errors, minimizes sample manipulation, and allows for efficient, high-throughput targeted re-sequencing with improved specificity and sensitivity, enabling the analysis of complex genomes with smaller sample sizes.
Implementation Method 1
hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide
Implementation Method 2
extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence
Implementation Method 3
amplifying the adaptor-modified product using bridge PCR to produce a PCR product
Data Source
Figure 1A
Figure 1B
Figure 1C~1D
AI summary
Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.