Locus-Specific Amplification on Solid Arrays
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Solution Overview
Problem
Next-generation sequencing technologies face challenges in efficiently targeting specific regions of interest due to random sampling methods, which result in redundant sequencing and inadequate representation of informative genomic regions.
Innovation Solution
The method involves solid-phase clonal amplification of hybridized target molecules on arrays, allowing for locus-specific targeting and reduction of redundant sequences by amplifying and analyzing specific DNA sequences using techniques like sequencing by synthesis or ligation, with adapters and hairpin oligonucleotides facilitating multiple copy generation and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If random sampling methods are used for sequencing, then genome coverage is achieved, but redundant sequencing occurs and informative regions are inadequately represented
Solution Approach 1:
The patent extracts and amplifies only the specific genomic regions of interest from the total genome using locus-specific probes. By capturing and selectively amplifying target regions (exon regions, disease-associated loci) while leaving non-target regions unamplified, the method eliminates redundant sequencing data and concentrates sequencing capacity on informative regions, thereby improving representation accuracy without wasting resources on redundant coverage.
Solution Approach 2:
The patent applies different treatment to different regions of the genome by using locus-specific probes that bind only to target regions. Each probe is designed with sequence specificity to capture particular genomic loci, creating local amplification zones where only relevant sequences are enriched. This local quality approach ensures that sequencing resources are concentrated on high-value regions while non-informative regions are excluded, resolving the contradiction between comprehensive coverage and redundant sequencing.
2Measurement precision
If solid phase clonal amplification is performed on arrays, then locus-specific targeting is achieved, but the number of hybridized molecules is lower than necessary for efficient sequencing
Solution Approach 1:
The patent performs preliminary clonal amplification of target DNA molecules on the array surface before sequencing. By using bridge PCR or emulsion PCR to amplify captured targets in place on the solid support, sufficient quantities of DNA are generated at each locus-specific site. This preliminary amplification step ensures that when sequencing reactions are initiated, there are adequate numbers of molecules present, resolving the contradiction between specific targeting and sufficient molecule quantity.
Solution Approach 2:
The patent creates multiple copies of the same target sequence at each array location through clonal amplification. Each captured DNA molecule serves as a template for generating numerous identical copies via PCR or emulsion PCR. These copies are then used as templates for sequencing reactions, ensuring that sufficient molecular material is available for efficient sequencing while maintaining locus-specific targeting. The copying process transforms single captured molecules into amplifiable clusters.
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 enables efficient, locus-specific amplification and sequencing of target regions, reducing redundancy and improving the accuracy and efficiency of DNA analysis by anchoring amplification and allowing for high-throughput, low-redundancy sequencing of genomic regions.
Implementation Method 1
hybridizing a primer to the adapter sequence and extend the primer
Implementation Method 2
extend the primer to make a copy of the extended target-specific probes
Implementation Method 3
repeating step (e) at least once, wherein a copy of the extended target-specific probe is displaced
Data Source
AI summary
Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.