Bead-Based Sequencing Using Phosphorothiolate Linkages
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Solution Overview
Problem
Current nucleic acid sequencing technologies face limitations in speed and accuracy due to the need for fragment separation, particularly through polyacrylamide gel electrophoresis, and struggle with the inefficiency of using reversible nucleotide terminators and the inaccuracies in sequencing methods like pyrosequencing and hybridization.
Innovation Solution
The development of new sequencing methods involving repeated cycles of duplex extension along a single-stranded template, ligation of labeled probes, and detection of labels, which eliminate the need for fragment separation and polymerase enzymes, utilizing scissile internucleosidic linkages and phosphorothiolate linkages for efficient sequencing, and employing solutions like SDS and sodium bisulfate to denature double-stranded nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If polyacrylamide gel electrophoresis is used for fragment separation, then sequencing accuracy is improved, but sequencing speed and productivity deteriorate
Solution Approach 1:
The invention extracts and eliminates the fragment separation step from the sequencing process. By using reversible terminators that can be chemically modified after incorporation, the method determines nucleotide identity through chemical transformation and detection rather than physical separation, thereby removing the productivity bottleneck while maintaining accuracy
Solution Approach 2:
The invention changes the detection parameter from physical separation based on fragment length to chemical detection based on nucleotide identity. By using fluorescently labeled reversible terminators and detecting their incorporation through fluorescence signals, the method transforms the sequencing detection mechanism to achieve both high speed and high accuracy
2Productivity
If reversible nucleotide terminators are used for sequencing, then sequencing speed is improved, but accuracy deteriorates due to inefficiency in terminator usage
Solution Approach 1:
The invention implements a feedback mechanism where the incorporation of reversible terminators is detected through fluorescence signals. The detection system provides real-time feedback on which nucleotides have been incorporated, allowing for accurate determination of nucleotide identity while maintaining high sequencing speed through efficient terminator usage
Solution Approach 2:
The invention replaces the mechanical/physical separation system with a chemical detection system. Instead of physically separating fragments by size, the method uses chemical modification of incorporated terminators and detects them through fluorescence, thereby improving both speed and accuracy simultaneously
3Extent of automation
If capillary electrophoresis is used for separation, then automation is improved, but sequencing speed and accuracy still deteriorate due to time-consuming separation and size-based discrimination
Solution Approach 1:
The invention extracts and eliminates the electrophoresis separation step entirely from the automated sequencing process. By using reversible terminators with fluorescent labels that can be detected directly after incorporation, the method removes the time-consuming separation bottleneck while maintaining automation through robotic liquid handling and automated fluorescence detection
Solution Approach 2:
The invention enables continuous sequencing by eliminating the interruption caused by separation steps. The reversible terminator method allows for continuous cycles of nucleotide incorporation, detection, and data collection without the need to pause for fragment separation, thereby dramatically improving sequencing speed while maintaining automation
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
These methods enable high-throughput sequencing with improved accuracy and efficiency, allowing for rapid determination of nucleotide sequences without the bottlenecks of traditional separation techniques and enzyme reliance, and provide effective solutions for sequence determination and error checking.
Implementation Method 1
employing solutions like SDS and sodium bisulfate to denature double-stranded nucleic acids
Data Source
AI summary
The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.