Cyclic Cleavable Nucleotides for Polymerase-Compatible DNA Sequencing
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Solution Overview
Problem
Existing DNA sequencing methods, particularly sequencing by synthesis (SBS), lack modified nucleotides that are effectively recognized by DNA polymerases and efficiently incorporated into growing DNA chains, hindering the goal of achieving the $1,000 genome.
Innovation Solution
Development of modified nucleotides with cleavable linkers and reversible terminators, including compounds with specific chemical moieties that are recognized by DNA polymerases and can be incorporated into DNA chains, allowing for accurate sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If modified nucleotides are designed with cleavable linkers and reversible terminators, then sequencing accuracy is improved, but nucleotide incorporation efficiency by DNA polymerase deteriorates
Solution Approach 1:
The patent modifies the chemical structure of nucleotides by changing parameters such as introducing cyclic cleavable moieties ( Rings A and B with specific heteroatom arrangements) and reversible terminator groups at the 3' position. These structural parameter changes enable the nucleotides to be recognized by DNA polymerase while maintaining sequencing accuracy through controlled incorporation and subsequent cleavage.
Solution Approach 2:
The invention creates composite nucleotide structures combining multiple functional elements: the nucleobase, sugar moiety, phosphate group, cleavable linker with cyclic structure (Rings A and B), and reversible terminator. This composite design integrates the benefits of each component to achieve both polymerase recognition and accurate sequencing through controlled cleavage.
2Measurement precision
If nucleotides with complex cyclic cleavable moieties are used, then sequencing specificity is improved, but ease of manufacture deteriorates
Solution Approach 1:
The cleavable linker is segmented into distinct functional regions: Ring A with two adjacent sulfur atoms, Ring B with specific heteroatom arrangement, and the cleavable bond between them. This segmentation allows for modular synthesis and independent optimization of each region's properties while maintaining overall specificity.
Solution Approach 2:
The cyclic cleavable moiety acts as an intermediary structure that temporarily remains attached to the nucleotide during synthesis and sequencing, then is cleaved to release the sequencing information. This intermediary function enables specificity without requiring complex manufacturing processes, as the cleavage step simplifies the final product.
Data Source
AI summary
Disclosed herein, inter alia, are methods and nucleotide compounds including cleavable moieties, and methods of use thereof.