Non-cleavable Terminating Nucleotides for Parallel DNA Sequencing
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Solution Overview
Problem
Current DNA sequencing technologies are expensive, labor-intensive, and time-consuming, making them unsuitable for broad application in human sequence variation studies, particularly in identifying rare and dispersed single nucleotide polymorphisms (SNPs) that underlie common diseases.
Innovation Solution
Development of nucleoside and nucleotide compounds with noncleavable groups labeled with fluorescent dyes that can efficiently terminate DNA synthesis, allowing for rapid and accurate sequencing of large oligomers in a parallel format, compatible with commercially available DNA polymerases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional Sanger sequencing methods are used, then accurate DNA sequencing can be achieved, but the process becomes expensive, labor-intensive, and time-consuming
Solution Approach 1:
The patent divides the DNA sequencing process into multiple parallel reactions, each containing all four dNTPs and one type of ddNTP analog with a specific fluorescent label. This segmentation allows simultaneous sequencing of multiple DNA fragments in parallel, dramatically increasing throughput while maintaining accuracy through the same chain termination mechanism
Solution Approach 2:
The patent modifies the chemical parameters of the terminating nucleotides by incorporating non-cleavable groups and fluorescent labels at specific positions (alpha, beta, or gamma). These parameter changes enable direct detection of terminated fragments without additional processing steps, improving both throughput and efficiency
2Measurement precision
If traditional Sanger sequencing methods are used, then accurate DNA sequencing can be achieved, but the process becomes expensive and labor-intensive
Solution Approach 1:
The patent combines multiple functions into a single reagent molecule: the terminating ddNTP analog incorporates both the chain-terminating function and the fluorescent detection function in one compound. This merging eliminates the need for separate labeling steps and reduces the number of reagents required, making the process more cost-effective
Solution Approach 2:
The fluorescent label is permanently attached to the terminating nucleotide analog, allowing the molecule to self-detect and self-report its incorporation into the DNA strand. This self-service capability eliminates the need for additional detection reagents or processing steps, reducing both cost and complexity
3Measurement precision
If traditional Sanger sequencing methods are used, then accurate DNA sequencing can be achieved, but the process becomes time-consuming
Solution Approach 1:
The patent enables continuous sequencing by using fluorescently labeled terminating nucleotides that can be detected in real-time during the polymerase reaction. The reaction and detection occur simultaneously without interruption, eliminating time-consuming post-reaction processing steps such as gel electrophoresis or additional labeling steps
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
The use of these compounds enables high-throughput, cost-effective DNA sequencing with termination efficiencies ranging from 70% to 100%, facilitating the identification of SNPs and other genetic information, thereby supporting more accurate disease diagnosis and targeted therapeutics.
Implementation Method 1
nucleoside and nucleotide compounds with noncleavable groups labeled with fluorescent dyes
Data Source
AI summary
Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a non-cleavable terminating group. The non-cleavable-fluorescent group is designed to terminate DNA synthesis so that DNA oligomers can be sequenced efficiently in a parallel format. These reagents and methods will lead to more accurate identification of polymorphisms and other valuable genetic information.