Sequencing Reagent Kit Using Disulfide Reduction to Remove Polymerase
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Residual DNA polymerase with strand displacement activity interferes with sequencing by entangling with chemically modified dNTPs, leading to increased background signal and reduced signal-to-noise ratio during multiple displacement amplification.
Innovation Solution
A kit comprising a disulfide-reducing agent to break disulfide bonds in DNA polymerase, a blocking reagent to irreversibly block thiol groups, and a protonating agent like ammonium salts to break hydrogen bonds, facilitating the removal of residual polymerase, thereby preventing entanglement with modified dNTPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If elution is performed to remove DNA polymerase, then sequencing quality should improve, but residual polymerase still entangles with modified dNTPs causing increased background signal
Solution Approach 1:
The patent extracts and removes the harmful DNA polymerase component through elution, but the problem is that complete removal is difficult. The residual polymerase remains and causes entanglement with modified dNTPs, leading to increased background signal. This principle is applied but incomplete, creating the technical contradiction.
Solution Approach 2:
The patent introduces an intermediary substance (not specified in the excerpt) that can bind to or neutralize the residual DNA polymerase, preventing its entanglement with modified dNTPs. This intermediary acts as a mediator between the polymerase and dNTPs, resolving the harmful interaction while maintaining sequencing quality.
2Object-affected harmful factors
If more elution reagent is used to remove residual polymerase, then background signal decreases, but elution time increases
Solution Approach 1:
The patent changes the parameters of the elution process, such as the composition, concentration, or pH of the elution reagent, to achieve more effective removal of residual DNA polymerase. By optimizing these parameters, the patent reduces background signal without requiring excessive elution time, resolving the contradiction between signal quality and process efficiency.
3Measurement precision
If elution time is extended to remove all polymerase, then signal-to-noise ratio improves, but sequencing efficiency decreases
Solution Approach 1:
The patent performs preliminary actions during the elution process, such as pre-treating the residual polymerase or preparing the elution reagent in advance, to enhance its removal efficiency. This preliminary action allows for more effective polymerase removal in a shorter time, improving signal-to-noise ratio without sacrificing sequencing efficiency.
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
Improves sequencing quality by reducing background signal and maintaining a high signal-to-noise ratio, while also reducing the amount of elution reagent required and shortening the elution time.
Implementation Method 1
the inventors innovatively first use a disulfide-reducing agent during the elution process to break these disulfide bonds in the enzyme structure
Implementation Method 2
then use a blocking reagent to block thiol groups, thereby allowing the dNTPs to be released
Implementation Method 3
By using a protonating agent during the elution process, the hydrogen bonds can be broken, facilitating the removal of DNA polymerase
Data Source
AI summary
Provided are a reagent kit and use thereof in sequencing. The reagent kit includes a first reagent and a second reagent. The first reagent is selected from a disulfide bond reducing agent. The second reagent is selected from a blocking reagent suited to blocking a disulfide bond.