DNA Sequencing by Polymerization Pause Detection
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Solution Overview
Problem
Current nucleic acid sequencing methods are costly due to the use of labeled nucleotides and require amplification steps, which are time-consuming and prone to errors, while also lacking sensitivity and being susceptible to contamination.
Innovation Solution
A method that determines the physical location of replication pauses in a nucleic acid sequence without the need for amplification, using physical techniques to measure the distance between the ends of a double-stranded nucleic acid molecule under tension, allowing for the sequencing of single molecules and parallel sequencing of multiple strands independently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If labeled nucleotides are used for sequencing, then sequencing can be performed, but costs increase significantly
Solution Approach 1:
The patent extracts and removes the labeling step from the sequencing process. Instead of using labeled nucleotides, the invention detects the physical location of replication pauses caused by natural nucleotide incorporation, thereby eliminating the need for expensive labels while maintaining sequencing capability
Solution Approach 2:
The invention uses simple, unlabeled nucleotides that can be incorporated and then discarded after serving their purpose of causing replication pauses. These disposable nucleotides are much cheaper than labeled alternatives and achieve the same sequencing function
2Quantity of substance
If amplification steps are used for sequencing, then sufficient material for sequencing is obtained, but time consumption increases and errors are introduced
Solution Approach 1:
The invention enables single molecules to serve themselves for sequencing without requiring amplification. Each individual nucleic acid molecule can be sequenced directly, eliminating the time-consuming amplification steps while maintaining sufficient signal for detection
Solution Approach 2:
The patent segments the sequencing process into independent single-molecule events rather than requiring bulk amplification. Multiple single molecules can be processed in parallel, reducing total sequencing time while avoiding amplification-related errors
3Quantity of substance
If amplification steps are used for sequencing, then sufficient material is obtained, but susceptibility to contamination increases
Solution Approach 1:
Single nucleic acid molecules perform the sequencing function independently without amplification, eliminating the contamination risks associated with amplification processes while maintaining sufficient material for detection through direct single-molecule analysis
4Quantity of substance
If physical techniques are used to measure replication pauses, then labeled nucleotides are eliminated, but measurement precision requirements increase
Solution Approach 1:
The patent uses optical detection methods to monitor physical changes in the nucleic acid molecule during replication. By detecting changes in light absorption or fluorescence as the replication fork pauses at different locations, the system achieves precise spatial resolution without requiring labeled nucleotides
Solution Approach 2:
The invention employs force spectroscopy or optical tweezers to detect mechanical changes in the nucleic acid molecule during replication. By measuring subtle mechanical signals associated with replication pauses, the system achieves high measurement precision through physical rather than chemical means
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 costs, eliminates the need for labeled nucleotides, improves sequencing accuracy, and enhances reliability by enabling multiple measurements on the same molecule, while allowing for the parallel sequencing of multiple strands with improved error rates and sensitivity.
Implementation Method 1
a) denaturing a double-stranded nucleic acid molecule corresponding to the nucleic acid sequence; b) hybridizing a single-stranded nucleic acid molecule ('the primer') with the denatured double-stranded nucleic acid molecule; c) applying a tension to the hybridized primer/double-stranded nucleic acid molecule obtained in b); d) incubating the hybridized primer/double-stranded nucleic acid molecule obtained in step b) with a polymerase in conditions which will lead to at least one pause in replication
Data Source
AI summary
Described herein is a method for determining a nucleic acid sequence, said method comprising: a) denaturing a double-stranded nucleic acid molecule corresponding to the said nucleic acid sequence; b) hybridizing a single-stranded nucleic acid molecule, the primer, with the said denatured double-stranded nucleic acid molecule; c) applying a tension to the hybridized primer/double-stranded nucleic acid molecule obtained in b); d) incubating the hybridized primer/double-stranded nucleic acid molecule obtained in b) with a polymerase in conditions which will lead to at least one pause in replication; and e) determining a position of the said pause in replication with respect to one end of the double-stranded nucleic acid.