Decorated Nucleic Acid Sequencing via Nicking and Gapping
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Solution Overview
Problem
Current sequencing technologies face challenges in accurately and cost-effectively analyzing long single nucleic acid molecules, particularly in distinguishing between the two copies of the human genome, due to limitations in providing contextual information and compatibility with clinical time and cost constraints.
Innovation Solution
A method involving the use of stretched decorated nucleic acids, where nucleic acids are nicked, gapped, and labeled with probes to create substrates that allow for optical resolution and sequence analysis, enabling accurate sequencing by determining the order and spacing of probes on the substrate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If long-read sequencing methods are used to preserve contextual integrity, then haplotype information is maintained, but the cost and time for clinical diagnostics become prohibitive
Solution Approach 1:
The patent segments the long nucleic acid molecule into multiple shorter readable portions by introducing nicks and gaps at specific intervals. This allows the molecule to be processed in manageable segments while preserving the overall contextual information through the spacing pattern of probes along the entire length of the original molecule.
Solution Approach 2:
The patent introduces probes as intermediary elements that bind to the nucleic acid at specific locations. These probes serve as mediators that enable detection and sequencing while maintaining the spatial relationships and contextual information of the original long molecule.
2Device complexity
If fixed array technologies are used to fragment the genome, then preparation complexity is reduced, but contextual information between the two genome copies is lost
Solution Approach 1:
The patent performs preliminary nicking and gap introduction on the long nucleic acid molecules before they are introduced into the fixed array system. This preliminary action prepares the molecules for efficient processing in the array while preserving their length and contextual information, combining the advantages of both approaches.
3Loss of information
If single molecule sequencing is used to provide contextual information, then haplotype integrity is maintained, but signal intensity is insufficient to overcome noise
Solution Approach 1:
The patent combines multiple probes along the length of a single nucleic acid molecule to create a composite signal. By merging the signals from multiple probes that are spatially distributed along the molecule, the overall signal intensity is amplified while maintaining the haplotype information encoded in the spacing pattern.
Solution Approach 2:
The patent changes the parameter of signal intensity by introducing multiple probes per molecule and using optical amplification methods. This increases the detectable signal from single molecules to levels that can overcome background noise while preserving the sequential information.
4Measurement precision
If redundant sequence analysis is performed to ensure accuracy, then sequencing precision is improved, but time and cost increase significantly
Solution Approach 1:
The patent adds the dimension of spatial information by utilizing the spacing and positioning of probes along the nucleic acid molecule. This spatial dimension provides additional information that enhances sequencing accuracy without requiring redundant analysis of the same sequence data multiple times.
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 accurate and cost-effective long-read sequencing, providing contextual integrity and haplotype information, overcoming the limitations of existing technologies in clinical diagnostics and genomic analysis.
Implementation Method 1
adding an exonuclease to the nicked nucleic acid to form a gapped nucleic acid
Implementation Method 2
adding a first set of labeled probes to the gapped nucleic acid such that at least one of the first set of labeled probes hybridizes to single stranded areas of said gapped nucleic acid
Implementation Method 3
adding a first set of recA invasive labeled probes to the double stranded nucleic acid to form D-loops within the double stranded nucleic acid
Implementation Method 4
the stretched decorated nucleic acids are positioned on the substrate in such a way that they are optically resolvable
Data Source
AI summary
The present invention provides a sequence interrogation chemistry that combines the accuracy and haplotype integrity of long-read sequencing with improved methods of preparing genomic nucleic acids and analyzing sequence information generated from those nucleic acids. The present invention encompasses compositions comprising decorated nucleic acids stretched on substrates. The present invention further encompasses methods of making stretched decorated nucleic acids and methods of using decorated nucleic acids to obtain sequence information.