Nucleic Acid Template Preparation via Controlled Strand Cleavage
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Solution Overview
Problem
Existing nucleic acid sequencing methods using immobilized double-stranded templates on solid supports, such as polyacrylamide hydrogels, face inefficiencies due to 'bridged' structures where both strands are attached, making it difficult to hybridize sequencing primers effectively, and reliance on restriction endonucleases for linearization is problematic due to unpredictable cleavage sites.
Innovation Solution
A method involving the cleavage of one or both strands of double-stranded nucleic acid molecules attached to a solid support, followed by denaturation to generate a partially or substantially single-stranded template, without using restriction or nicking endonucleases, using pre-determined cleavage sites or non-enzymatic chemical methods like periodate treatment or uracil DNA glycosylase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If both strands of double-stranded nucleic acid are attached to solid support forming bridged structures, then immobilization efficiency is improved, but primer hybridization efficiency deteriorates
Solution Approach 1:
The double-stranded nucleic acid is segmented into separate strands through controlled cleavage. One strand remains immobilized on the solid support while the other strand is released, creating a configuration where the immobilized strand serves as template for primer hybridization without the interference of the second immobilized strand
Solution Approach 2:
One strand of the double-stranded nucleic acid is extracted from the bridged structure through selective cleavage and removal. This leaves only the necessary immobilized strand for sequencing while eliminating the problematic second strand that prevented effective primer hybridization
2Ease of manufacture
If restriction endonucleases are used for linearization, then template preparation is achieved, but cleavage precision deteriorates due to unpredictable cleavage sites
Solution Approach 1:
A specific intermediary enzyme (such as uracil-DNA glycosylase or apurinic/apyrimidinic endonuclease) is introduced that recognizes and cleaves only at the modified base site. This intermediary provides precise and predictable cleavage at the intended location, eliminating the unpredictable multiple cleavage sites problem associated with restriction endonucleases
Solution Approach 2:
The nucleic acid template is modified by incorporating non-natural bases or chemical modifications at specific positions. These parameter changes create unique recognition sites for selective cleavage enzymes, enabling precise control over where cleavage occurs rather than relying on natural restriction sites
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 enhances the efficiency of nucleic acid sequencing by creating suitable templates for hybridization and sequencing, while avoiding the limitations of enzyme-dependent linearization methods, allowing for controlled and predictable template preparation compatible with hydrogel surfaces.
Implementation Method 1
The diol linkage is cleaved by treatment with periodate
Implementation Method 2
uracil DNA glycosylase
Data Source
AI summary
The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise: providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end, cleaving one or both strands of the double-stranded nucleic acid molecule, and subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction.