Nucleic Acid Sequencing via Closed Complex Trapping
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Solution Overview
Problem
Current pyrosequencing technologies face challenges with non-specificity and accuracy, particularly when sequencing stretches of consecutive nucleotides with the same base, due to background noise and increased light emission, which affects the precision of determining nucleotide lengths.
Innovation Solution
The method involves forming a closed complex with the DNA polymerase and nucleotide during DNA synthesis, trapping the complementary nucleotide, and using divalent metal ions to release pyrophosphate for detection, allowing for accurate identification of each nucleotide without the need for nucleotide labeling, and can be applied in parallel sequencing using microbeads and picoliter-sized reaction wells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If pyrosequencing is used to sequence stretches of consecutive nucleotides with the same base, then throughput is improved, but measurement precision deteriorates due to increased light emission and background noise
Solution Approach 1:
The patent segments the sequencing process into discrete cycles, where in each cycle only one nucleotide type is added to the reaction mixture. This allows the polymerase to process one base at a time, preventing the accumulation of light emission signals that occurs when multiple nucleotides are present simultaneously. Each cycle produces a distinct, measurable signal corresponding to a single nucleotide incorporation event.
Solution Approach 2:
The patent implements periodic action by cycling through the addition of different nucleotide types in repeated cycles. Each cycle involves adding a specific nucleotide, allowing polymerase binding and PPi release, then removing that nucleotide before adding the next type. This periodic process ensures that light emission signals are generated discretely and can be precisely measured for each nucleotide position.
2Speed
If conventional pyrosequencing is used, then sequencing speed is improved, but reliability deteriorates due to non-specificity and background noise
Solution Approach 1:
The patent segments the nucleotide addition process into separate cycles for each nucleotide type (dATP, dCTP, dGTP, dTTP). By adding only one nucleotide type per cycle, the system eliminates non-specific binding and background noise that would occur if all nucleotides were present simultaneously. This segmentation ensures that only the correct nucleotide can bind to the polymerase at any given time, improving reliability.
Solution Approach 2:
The patent performs preliminary removal of the previously added nucleotide before adding the next nucleotide type. This preliminary action ensures that the reaction mixture contains only the next nucleotide to be added, preventing non-specific reactions and background noise. The removal step prepares the reaction system cleanly for the next specific nucleotide addition, maintaining high reliability throughout the sequencing process.
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 eliminates non-specificity, provides accurate sequencing for each base, and enables ultra-high throughput, non-ambiguous nucleic acid sequencing by freezing polymerase activity and using sensitive detection methods, even for single molecules or collections of identical sequences.
Implementation Method 1
the DNA polymerase-DNA complexes formed are known to undergo a rate-limiting, conformational transition from an 'open' to 'closed' state, upon binding of the correct dNTP or ddNTP at the active site
Implementation Method 2
the production of PPi reveals the identity of the next correct base
Implementation Method 3
Luciferase uses the ATP to convert luciferin to oxyluciferin, emitting a photon
Data Source
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AI summary
This invention provides methods for massive parallel nucleic acid analysis. A closed complex of nucleic acid template, nucleotide and polymerase can be formed during polymerase reaction, absent divalent metal ion. This is used to trap the nucleotide complementary to the next template nucleotide in the closed complex. Detection of the trapped nucleotide allows determination of the sequence of this next correct nucleotide. In this way, sequential nucleotides of a nucleic acid template can be identified, effectively determining the sequence. This method is applied to sequence multiple templates in parallel, particularly if they are immobilized on a solid support.