Rapid Nucleic Acid Hybridization via Thermal Cycling
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Solution Overview
Problem
Current targeted next-generation sequencing technologies face significant bottlenecks in the time-consuming and costly process of selectively capturing and enriching targeted exons or intron regions scattered over genomic, mitochondria, and other forms of DNA, with conventional hybrid capture procedures often taking 16 hours to over 70 hours.
Innovation Solution
A novel hybridization method using a composition with a divalent cation salt concentration of 100 mM to 600 mM, a buffering agent, and a volume-excluding/thickening agent like hydroxypropyl methyl cellulose, allowing for rapid nucleic acid hybridization by incubating at two different temperatures, significantly reducing the enrichment time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If conventional hybrid capture procedures are used for target enrichment, then enrichment completeness is achieved, but the process takes 16 hours to over 70 hours
Solution Approach 1:
The patent applies parameter changes by using a two-temperature cycling incubation protocol (alternating between 65°C and 37°C) instead of conventional constant temperature incubation. This temperature cycling, combined with high divalent cation concentration (100-600 mM), accelerates hybridization kinetics while maintaining enrichment completeness, reducing the process from 16-70 hours to approximately 1 hour.
Solution Approach 2:
The invention employs periodic action through two-temperature cycling incubation, where the hybridization mixture is repeatedly subjected to alternating high and low temperatures. This periodic thermal treatment enhances the hybridization rate and target enrichment efficiency, achieving complete enrichment in about 1 hour compared to the conventional 16-70 hour timeframe.
2Productivity
If high divalent cation concentration is used in the hybridization composition, then hybridization speed increases, but non-specific binding may increase
Solution Approach 1:
The patent utilizes parameter changes by implementing two-temperature cycling incubation in conjunction with high divalent cation concentration (100-600 mM). The alternating temperature protocol enhances hybridization kinetics to achieve rapid enrichment while the periodic thermal treatment also helps maintain specificity by allowing proper strand separation and reannealing, preventing non-specific binding despite the high cation concentration.
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 enables rapid target enrichment of genomic DNA, completing the hybridization process in as little as 1 hour, thereby speeding up the entire targeted next-generation sequencing process and improving efficiency and cost-effectiveness.
Implementation Method 1
a method for nucleic acid hybridization of a target nucleic acid and a bait nucleic acid, comprising contacting a target nucleic acid and a bait nucleic acid with a composition to form a hybridization mixture
Implementation Method 2
a volume-excluding/thickening agent that has a concentration in a range from 0.002% to 0.1%, and wherein the volume-excluding/thickening agent is selected from the group consisting of hydroxypropyl methyl cellulose (HPMC) and methyl cellulose
Implementation Method 3
incubating the mixture at two hybridization temperatures, which can be different or the same
Data Source
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AI summary
The present invention relates to compositions and methods of target enrichment or selection of nucleic acids using hybridization, which can be used in, e.g., next-generation sequencing.