Nucleotide Sequencing with Selective Cleaving Activity
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Solution Overview
Problem
Current nucleic acid sequencing methods, such as single-molecule sequencing-by-synthesis, face challenges with errors in base calling due to incorrect nucleotide incorporation and identification, leading to inaccuracies in determining the sequence of nucleic acid templates.
Innovation Solution
The method involves a sequencing mixture with a polymerase enzyme, a template nucleic acid, and nucleotide analogs with labeled leaving groups, where a selective cleaving activity differentiates incorporated from unincorporated nucleotides by cleaving the labeled leaving group more effectively after incorporation, allowing for accurate sequence determination through light detection during the polymerase reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single-molecule sequencing-by-synthesis is used, then sequencing throughput and speed are improved, but sequencing accuracy deteriorates due to errors in base calling
Solution Approach 1:
The sequencing method segments the detection process into two distinct chemistry steps: (1) incorporation of nucleotide analogs with labeled leaving groups, and (2) selective cleavage of the leaving group. This segmentation allows separate optimization of signal generation and signal detection, improving both throughput and accuracy by eliminating interference from unincorporated nucleotides during the detection phase.
Solution Approach 2:
The patent introduces a labeled leaving group as an intermediary component attached to the nucleotide analog. This intermediary serves as a temporary carrier of the label that is selectively removed after incorporation, allowing the label to signal successful incorporation without remaining on the DNA strand to cause interference in subsequent cycles, thus improving base-calling accuracy.
2Measurement precision
If labeled leaving groups are used on nucleotide analogs, then sequencing accuracy is improved through distinct signals, but device complexity increases due to additional chemistry steps
Solution Approach 1:
The system employs a self-service mechanism where the polymerase enzyme itself performs the selective cleavage of the labeled leaving group after nucleotide incorporation. The polymerase's natural exonuclease activity is harnessed to remove the leaving group, eliminating the need for separate external enzymes or complex additional reagents, thus reducing overall system complexity while maintaining high sequencing accuracy.
Solution Approach 2:
The patent utilizes parameter changes in the chemical environment to achieve selective cleavage. By adjusting conditions such as pH or adding specific buffers that activate the polymerase's exonuclease activity, the system triggers leaving group removal only under controlled conditions after incorporation, simplifying the process by using inherent enzyme properties rather than requiring separate enzymatic systems.
3Measurement precision
If selective cleavage activity is introduced to differentiate incorporated from unincorporated nucleotides, then measurement precision is improved, but loss of time occurs due to additional cleavage step
Solution Approach 1:
The patent merges the cleavage function with the polymerase enzyme by utilizing its inherent exonuclease activity. This combining of incorporation and cleavage functions into a single enzyme eliminates the need for separate addition of external enzymes and their associated incubation times, thereby reducing the time loss while maintaining the precision benefits of selective leaving group removal.
Solution Approach 2:
The system maintains continuous useful action by having the polymerase perform both nucleotide incorporation and subsequent leaving group cleavage in a continuous manner without requiring removal of the enzyme or addition of separate reagents between steps. This continuity eliminates idle time and maintains the polymerase in productive action throughout the sequencing cycle.
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 accuracy by providing distinct signals for incorporated nucleotides, reducing errors from branching or non-cognate events, and allowing for higher confidence in determining the nucleic acid sequence.
Implementation Method 1
a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog
Implementation Method 2
carrying out template directed synthesis of a growing nucleic acid strand
Implementation Method 3
providing a sequencing mixture comprising a polymerase enzyme
Implementation Method 4
detecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid
Data Source
AI summary
Methods, Compositions, and Systems are provided for nucleic acid sequencing where the sequential incorporation of nucleotides uses two distinct chemical steps. A plurality of nucleotide analogs, each having a labeled leaving group at its 3′ hydroxyl can be sequentially added to a growing strand in the presence of a selective cleaving activity that cleaves the 3′ hydroxyl leaving group preferentially after it has been incorporated. The selective cleaving agent can comprise an exonuclease activity, and the exonuclease activity can be a polymerase-associated exonuclease activity. Nucleotide analogs having labels on both a cleavable polyphosphate portion and on a 3′ hydroxyl leaving group can provide signals characteristic of nucleotide analog incorporation. Systems having illumination optics, collection optics, and substrates observe signals from the labels as they are being incorporated into a growing nucleic acid strand, allowing for the sequencing of template nucleic acids.