Ion Sensor DNA Sequencing with Reversible Terminators
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Solution Overview
Problem
Current high-throughput sequencing technologies face challenges in accurately decoding homopolymer stretches longer than 4-5 bases and suffer from false positives due to spontaneous decomposition of natural nucleotides, leading to high costs and inefficiencies in achieving the goal of $1,000 genome sequencing with low error rates.
Innovation Solution
A method using deoxyribonucleotide or ribonucleotide triphosphate analogues with hydrocarbyl or substituted hydrocarbyl blocking groups, photochemically cleavable with a mass less than 300 daltons, which are incorporated into DNA or RNA sequences, allowing for precise detection of nucleotide incorporation by hydrogen ion concentration changes and subsequent cleavage to enable accurate sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If natural nucleotides are used in ion sensor sequencing, then the sequencing process can proceed, but accurate decoding of homopolymer stretches longer than 4-5 bases becomes difficult
Solution Approach 1:
The nucleotide analogues are segmented into four distinct types, each carrying a unique mass tag (different heavy atom compositions). This segmentation allows the ion sensor to distinguish between different nucleotides incorporated during homopolymer stretches by detecting the specific mass of each incorporated nucleotide type, thereby resolving the ambiguity in decoding long homopolymer regions.
Solution Approach 2:
Each nucleotide analogue is equipped with a locally distinct heavy atom composition (different mass tags) at a specific position in the molecule. This local quality difference enables the ion sensor to identify the specific nucleotide type incorporated, even when multiple identical nucleotides are added in sequence during homopolymer decoding, thus improving measurement precision without compromising reliability.
2Productivity
If natural nucleotides are used, then sequencing can be performed, but false positives occur due to spontaneous decomposition of free dNTPs
Solution Approach 1:
The harmful property of spontaneous decomposition is extracted and eliminated by replacing natural nucleotides with synthetic nucleotide analogues containing stable heavy atom compositions. These analogues do not undergo spontaneous decomposition, thereby removing the source of false positives while maintaining sequencing productivity.
Solution Approach 2:
The nucleotide analogues are designed as disposable reagents with built-in stability features. Each analogue incorporates a stable heavy atom composition that prevents spontaneous decomposition, ensuring that each sequencing cycle produces reliable signals without generating false positives, thus improving the error rate while maintaining throughput.
3Measurement precision
If base-by-base addition strategy is used, then each nucleotide can be identified, but the process becomes time-consuming and costly
Solution Approach 1:
The nucleotide analogues enable continuous sequencing by allowing the ion sensor to detect multiple nucleotide incorporations in sequence without requiring interruption for signal differentiation. The unique mass tags allow continuous detection of each incorporated nucleotide type, maintaining measurement precision while reducing the time loss associated with repeated base-by-base addition cycles.
4Reliability
If conventional sequencing methods are used, then sequencing can be performed, but the cost per genome remains prohibitively high
Solution Approach 1:
The invention changes the physical parameter of nucleotide detection from indirect methods (fluorescence, light production) to direct mass detection using ion sensors. This parameter change enables more efficient use of reagents and faster sequencing cycles, reducing the quantity of substances required per genome sequence while maintaining high accuracy, thereby lowering the cost per genome.
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 sequencing by overcoming the limitations of homopolymer decoding and false positives, achieving precise nucleotide identification and reducing reagent costs, thereby moving towards the goal of $1,000 genome sequencing with improved accuracy and efficiency.
Implementation Method 1
contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and a deoxyribonucleotide triphosphate (dNTP) analogue base under conditions permitting the DNA polymerase to catalyze incorporation of the dNTP analogue into the primer
Implementation Method 2
determining whether incorporation of the dNTP analogue has occurred in step (a) by detecting an increase in hydrogen ion concentration of the solution
Implementation Method 3
subsequently treating the incorporated dNTP analogue with light to photochemically cleave the R' group so as to replace the R' group thereof with an H atom thereby providing a 3' OH group at the 3' terminal of the DNA extension product
Data Source
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AI summary
This disclosure is related to a method for determining the identity of a nucleotide residue of a single- stranded DNA or RNA, or sequencing DNA or RNA, in a solution using an ion-sensing field effect transistor and reversible nucleotide terminators.