DNA Sequencing with 3' Unprotected Labeled Nucleotides
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Solution Overview
Problem
Current DNA sequencing methods, particularly DNA sequencing-by-synthesis (SBS), are slow and costly due to the need for modified DNA polymerases to incorporate chemically modified nucleotides and the requirement for high temperatures, which are inefficient and prone to phasing issues.
Innovation Solution
A method using non-capped fluorescently labeled nucleotides is employed, allowing the use of regular polymerases and eliminating the need for high temperatures, by incorporating nucleotides with cleavable linkers and removing fluorescent dyes after each cycle, enabling faster sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemically modified nucleotides with 3' capping groups are used for sequencing-by-synthesis, then the nucleotides can be incorporated into the growing DNA strand, but the process becomes slow requiring multiple steps including de-protection between each cycle
Solution Approach 1:
The invention extracts and removes the 3' capping group from the nucleotide structure, allowing the nucleotide to be incorporated without requiring subsequent de-protection steps. This extraction of the protective group eliminates a major bottleneck in the sequencing process and enables continuous rapid cycling.
Solution Approach 2:
Instead of protecting the 3' position and then removing protection after incorporation, the invention inverts the approach by using nucleotides that are inherently unprotected at the 3' position, allowing direct incorporation without protective groups. This inversion fundamentally changes the chemistry from protected-to-unprotected to unprotected-direct-incorporation.
2Productivity
If modified DNA polymerases are used to tolerate extensive modifications including 3' capping groups, then nucleotide incorporation is enabled, but the requirement for high temperatures and specialized enzymes increases cost and reduces efficiency
Solution Approach 1:
The invention changes the chemical parameters of the nucleotide by removing the 3' capping group, which fundamentally alters how the nucleotide interacts with the polymerase active site. This parameter change allows standard polymerases to incorporate the nucleotide without requiring modified enzymes or elevated temperatures.
3Measurement precision
If fluorescent dyes are attached to nucleotides via cleavable linkers for detection, then sequencing information can be obtained, but the linkers interfere with polymerase recognition and incorporation
Solution Approach 1:
The invention applies local quality by placing the fluorescent dye label specifically at the 5-position of the pyrimidine base or 7-position of the purine base, which are locations distant from the polymerase active site. This localized labeling maintains detection capability while avoiding interference with incorporation.
Solution Approach 2:
The invention uses a flexible linker as an intermediary between the fluorescent dye and the nucleotide base. This intermediary allows the dye to be positioned away from the active site while still enabling fluorescence detection, effectively mediating between the detection requirement and the incorporation efficiency.
4Measurement precision
If washing steps are performed after each nucleotide incorporation to remove unincorporated nucleotides, then signal accuracy is improved, but the overall sequencing time increases significantly
Solution Approach 1:
The invention merges the detection and washing steps by performing detection in the presence of unincorporated nucleotides using spatially resolved detection methods. This merging eliminates the need for separate washing steps between detection cycles, dramatically reducing the time per 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 reduces sequencing time and cost by using regular polymerases and minimizing phasing errors, achieving sequencing in a fraction of the time of traditional methods.
Implementation Method 1
detecting the incorporated nucleotides by excitation and detecting of the fluorescent emission radiation of the fluorescent dyes
Implementation Method 2
removing the fluorescent dyes from the incorporated nucleotides by cleaving the cleavable linkers
Data Source
Figure 1~2
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AI summary
DNA sequencing by sequential addition/incorporation of non 3'capped and fluorescently labeled nucleotides. Each incorporation of individual nucleotides (A, or T or G or C) are separated by a wash. After all incorporation using all four bases, the substrate is imaged then the dyes are cleaved, and the following cycle of incorporations, washes, imaging and cleave is resumed.