Multivalent Molecule Surface Chemistry for Pairwise Sequencing
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Solution Overview
Problem
Current polynucleotide sequencing technologies face limitations in throughput and signal-to-noise ratio due to inadequate surface chemistry and on-support amplification, leading to increased costs.
Innovation Solution
The method involves immobilizing single-stranded nucleic acid concatemer template molecules on a support, using multivalent molecules with specific core, spacer, and nucleotide units for sequencing, and conducting primer extension reactions to generate extended strands, which are then sequenced to improve sequencing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional sequencing methods are used, then sequencing can be performed, but throughput is limited and signal-to-noise ratio is poor
Solution Approach 1:
The invention segments the sequencing process into two separate directions (forward and reverse) using bidirectional sequencing. Multiple primers are used to sequence the template molecule from both 5' to 3' and 3' to 5' directions, allowing independent optimization of each sequencing reaction and improving overall throughput and signal quality
Solution Approach 2:
The invention transitions from unidirectional to bidirectional sequencing by adding a temporal and directional dimension to the sequencing process. By conducting sequencing reactions in both directions and combining the results, the system achieves higher throughput and better signal-to-noise ratio than single-direction methods
2Productivity
If conventional surface chemistry is used, then sequencing can proceed, but amplification efficiency is insufficient leading to increased costs
Solution Approach 1:
The invention employs composite surface chemistry structures combining solid support surfaces with immobilized primers and multivalent molecules. This composite approach enhances amplification efficiency by providing optimized binding sites while reducing reagent consumption and costs through reusable solid-phase systems
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 enhances sequencing efficiency and reduces costs by improving surface chemistry and amplification processes, leading to better throughput and signal quality.
Implementation Method 1
individual multivalent molecules in the first plurality of multivalent molecules of step (b) and in the second plurality of multivalent molecules of step (e) comprise (i) a core; and (ii) a plurality of nucleotide arms which comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit
Implementation Method 2
wherein the spacer is attached to the linker
Implementation Method 3
wherein the linker is attached to the nucleotide unit
Data Source
AI summary
The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.