Sequencing Wash Composition for Residual Signal and Phasing Control
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Solution Overview
Problem
Existing polynucleotide sequencing methods face issues with residual signal interference due to unincorporated nucleotides, leading to noise and inaccuracies in identifying subsequent nucleotides, and challenges in reducing phasing and error rates.
Innovation Solution
Incorporating a polymerase in a wash composition to facilitate the removal of residual signal and polymerase from the copy strand, thereby reducing residual signal, phasing, pre-phasing, and error rates, and potentially shortening sequencing cycle time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a wash step is performed to remove unincorporated detectable nucleotide, then residual signal is reduced, but sequencing cycle time increases
Solution Approach 1:
The patent modifies the chemical composition of the wash buffer by adding specific reagents (e.g., formamide, betaine, DMSO) to enhance the removal of residual nucleotides and polymerase, thereby reducing residual signal without requiring extended wash times, thus maintaining fast sequencing cycle times while improving measurement precision
Solution Approach 2:
The wash buffer acts as an intermediary composition that facilitates the removal of both unincorporated nucleotides and polymerase from the template-primer complex. The optimized buffer composition mediates between the need for thorough cleaning (to reduce residual signal) and the need for rapid processing (to maintain short cycle times)
2Reliability
If polymerase is retained on the copy strand after de-blocking, then residual signal increases, but sequencing accuracy decreases
Solution Approach 1:
The patent specifically targets and extracts the polymerase from the template-primer complex during the wash step. The optimized wash buffer composition promotes the dissociation and removal of polymerase along with unincorporated nucleotides, eliminating the source of residual signal while preserving the incorporated nucleotide signal for accurate sequencing
Solution Approach 2:
The patent converts the potentially harmful effect of polymerase retention (which causes residual signal) into a beneficial washing process. By designing the wash buffer to specifically promote polymerase release and removal, the previously problematic polymerase retention is transformed into an efficient cleanup mechanism that improves sequencing accuracy
3Productivity
If sequencing cycle time is shortened, then productivity increases, but phasing and error rate increase
Solution Approach 1:
The patent performs preliminary removal of unincorporated nucleotides and polymerase during the wash step that occurs early in the sequencing cycle. By thoroughly clearing residual materials before the next incorporation step begins, the system maintains high sequencing accuracy and reduces phasing errors even when cycle times are shortened, thereby enabling faster productivity without sacrificing precision
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 a polymerase in the wash composition effectively reduces residual signal, improves sequencing metrics, and allows for shorter sequencing cycles without compromising accuracy.
Implementation Method 1
employing a wash composition that includes a polymerase. Surprisingly, it has been found that employing a polymerase in a wash composition reduces residual signal following a de-block step
Implementation Method 2
Sequencing of a template polynucleotide strand may occur through multiple cycles of steps by which one detectable nucleotide per cycle is incorporated into a copy strand complementary to the template strand
Data Source
AI summary
A polynucleotide sequencing method includes a wash step that employs a composition including a polymerase. The composition may also include a plurality of nucleotides. The composition may be configured to prevent the polymerase from incorporating one of the plurality of nucleotides into a copy polynucleotide strand. The composition may be substantially free of Mg2+