Structure-Assembly Sequencing to Minimize Hybridization Bias
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing sequencing methods face challenges in accurately and efficiently determining the sequence of nucleotides in nucleic acids, particularly due to biases in oligonucleotide probe hybridization and ligation processes.
Innovation Solution
The use of template non-hybridizing nucleic acid structures in oligonucleotide probes, which include detectable moieties, allows for precise identification of nucleotides through hybridization and ligation cycles, reducing bias by ensuring single ligation events and enabling detection of multiple nucleotides simultaneously.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional oligonucleotide probes are used for sequencing, then hybridization can occur, but bias in hybridization and ligation processes reduces sequencing accuracy
Solution Approach 1:
The invention extracts and removes the template-binding portion of the oligonucleotide probe from the hybridization process. By using probes that bind to a template-independent structure (such as a hairpin or stem-loop) rather than directly to the template nucleic acid, the method eliminates hybridization bias that occurs when probes compete for template binding sites. This separation allows unbiased detection of multiple nucleotides simultaneously.
Solution Approach 2:
The invention introduces an intermediary structure (template non-hybridizing nucleic acid forming a stem-loop or hairpin) that mediates between the probe and the template. The probe binds to this intermediary structure rather than directly to the template, allowing the template sequence to be detected without the probe competing for template binding. This intermediary eliminates the harmful interaction between probe and template that causes hybridization bias.
2Productivity
If multiple nucleotides are detected in a single ligation cycle, then sequencing efficiency increases, but ensuring single ligation events without bias becomes more difficult
Solution Approach 1:
The invention segments the probe structure into distinct functional regions: a template-independent binding region (stem-loop) and a ligation region with the detectable nucleotide. This segmentation allows the probe to undergo a single, well-defined ligation event at the 3' end while the detectable moiety remains associated with the probe structure. The segmented design ensures that even though multiple probes are present, each undergoes independent single ligation events, maintaining ligation accuracy while enabling parallel detection of multiple nucleotides.
Solution Approach 2:
The invention uses multiple copies of the same probe structure (with different detectable nucleotides) that all bind to the same template-independent structure. This copying approach allows simultaneous detection of multiple nucleotides through parallel ligation events, each following the same reliable single-ligation mechanism. The standardized copied probe design ensures consistent ligation behavior across all probe types.
3Measurement precision
If template non-hybridizing nucleic acid structures are used in probes, then hybridization bias is reduced, but probe design and complexity increase
Solution Approach 1:
The invention applies local quality by creating a specific structural feature (stem-loop or hairpin) at a localized region of the probe that forms the template-independent binding site. The rest of the probe maintains its simple nucleotide sequence with a detectable moiety. This localized structural complexity only where needed allows the probe to achieve template-independent binding without requiring complete redesign of the entire probe structure, thus limiting the increase in overall complexity.
Solution Approach 2:
The invention changes the physical-chemical parameters of the probe by forming a stem-loop structure that creates a stable template-independent binding site. This structural parameter change allows the probe to bind to itself rather than to the template, eliminating hybridization bias. The stem-loop formation is achieved through standard nucleic acid base-pairing rules, requiring only moderate changes to probe design while achieving the desired functional parameter change of template-independent 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
This approach enhances the accuracy and efficiency of nucleotide sequencing by minimizing hybridization bias and allowing for rapid identification of nucleotide sequences along a nucleic acid template.
Implementation Method 1
repeated cycles of duplex extension along a nucleic acid template, such as a single stranded nucleic acid template, using probes that facilitate detection of one or more or all of the nucleotides in an oligonucleotide probe that is hybridized and/or ligated in duplex extension to the nucleic acid template
Implementation Method 2
repeated cycles of duplex extension along a nucleic acid template, such as a single stranded nucleic acid template, using probes that facilitate detection of one or more or all of the nucleotides in an oligonucleotide probe that is hybridized and/or ligated in duplex extension to the nucleic acid template
Data Source
AI summary
A method of sequencing nucleic acids is provided using sequencing by ligation and/or sequencing by hybridization.