Nucleic Acid Sequencing with Non-Hybridizing Probe Structures
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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 hybridization and ligation processes.
Innovation Solution
The method employs sequencing by ligation and hybridization using probes with template non-hybridizing nucleic acid structures that include detectable moieties, allowing for the identification of nucleotides through repeated cycles of duplex extension in either the 5' to 3' or 3' to 5' direction, or in parallel, using different detectable moieties to differentiate between oligonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing methods are used, then sequencing can be performed, but biases in hybridization and ligation processes reduce accuracy
Solution Approach 1:
The patent introduces a template non-hybridizing nucleic acid structure as an intermediary element that does not bind to the template strand. This structure serves as a mediator to carry detectable moieties that enable nucleotide identification without participating in hybridization, thereby eliminating hybridization bias and improving measurement precision while maintaining reliability
Solution Approach 2:
The oligonucleotide probe is segmented into distinct functional regions: a template-hybridizing region for specific binding and a template non-hybridizing region for carrying detectable moieties. This segmentation allows the probe to perform hybridization and detection functions separately, eliminating the bias that occurs when these functions are combined in traditional sequencing methods
2Loss of information
If repeated cycles of duplex extension are performed, then complete sequencing is achieved, but process complexity increases
Solution Approach 1:
The template non-hybridizing nucleic acid structure serves multiple functions simultaneously: it prevents template binding, carries detectable moieties for nucleotide identification, and enables repeated ligation cycles. This multi-functionality reduces the need for separate components and simplifies the overall sequencing process while achieving complete sequence coverage
3Measurement precision
If detectable moieties are attached to probes, then nucleotide identification is enabled, but probe complexity increases
Solution Approach 1:
The detectable moiety function is extracted from the template-hybridizing region and placed in a separate template non-hybridizing region. This extraction allows the probe to maintain simple hybridization functionality while adding detection capability through the attached detectable moiety, reducing overall probe complexity while enabling precise nucleotide identification
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 bias and enhances the accuracy of nucleotide identification by ensuring fixed hybridization and ligation events, enabling the sequencing of entire nucleic acid sequences with high precision.
Implementation Method 1
Certain aspects include 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
The template non-hybridizing nucleic acid may include a detectable moiety corresponding to a known nucleotide in the oligonucleotide probe. The detectable moiety is detected. The template non-hybridizing nucleic acid may include a probe hybridization site corresponding to a known nucleotide in the oligonucleotide probe. A probe with a detectable moiety is then hybridized to the probe hybridization site and the detectable moiety is detected
Data Source
AI summary
A method of sequencing nucleic acids is provided using sequencing by ligation and/or sequencing by hybridization.