Sequencing Kit Sulfhydryl Blocking for Lower Background Signal
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Solution Overview
Problem
The residual phi29 DNA polymerase during second-strand sequencing entangles with dNTPs, leading to increased background signal and decreased signal-to-noise ratio due to non-specific adsorption, which affects sequencing quality.
Innovation Solution
A sequencing kit containing a sulfhydryl blocking reagent, such as N-ethylmaleimide or iodoacetamide, is used to break disulfide bonds in enzyme proteins, irreversibly blocking sulfhydryl groups to release fluorescent dyes and improve sequencing quality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If elution solvent is used to remove non-specifically bound fluorescent substances, then background signal reduction is attempted, but sequencing quality deteriorates due to incomplete removal and damage to sequencing efficiency
Solution Approach 1:
The patent applies preliminary action by blocking the sulfhydryl groups of phi29 DNA polymerase with iodoacetamide or N-ethylmaleimide before the sequencing process begins. This pre-treatment prevents the enzyme from non-specifically adsorbing fluorescent dyes during second-strand sequencing, thereby reducing background signal while maintaining sequencing quality without needing aggressive elution treatments that could damage the sequencing process
2Object-affected harmful factors
If elution concentration and time are increased to remove non-specific adsorption, then fluorescent substance removal is improved, but sequencing efficiency deteriorates
Solution Approach 1:
The patent performs the blocking treatment beforehand, so that when second-strand sequencing occurs, the polymerase is already blocked and cannot adsorb fluorescent dyes non-specifically. This eliminates the need for high-concentration or prolonged elution treatments, thereby maintaining high sequencing efficiency while effectively removing non-specific adsorption issues
Solution Approach 2:
The patent converts the potentially harmful non-specific adsorption property of phi29 DNA polymerase into a beneficial controlled interaction. By selectively blocking only the sulfhydryl groups involved in non-specific adsorption while leaving the catalytic activity intact, the enzyme's binding capability is redirected toward productive sequencing reactions rather than harmful background noise
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 sulfhydryl blocking reagent effectively reduces background signal and enhances the signal-to-noise ratio, improving sequencing quality by preventing polymerase impact during multiplex displacement amplification.
Implementation Method 1
first opening a disulfide bond in an enzyme protein structure such that the disulfide bond becomes two exposed sulfhydryl groups
Implementation Method 2
halogen substitution reaction, commonly using iodoacetamide
Implementation Method 3
addition reaction, commonly using N-ethylmaleimide (NEM)... Michael addition refers to a conjugated addition reaction
Data Source
AI summary
Provided are a sequencing kit containing a sulfydryl blocking reagent and an application of the sequencing kit. Also disclosed is a method for reducing a sequencing background signal. The method comprises the following steps: (1) opening a three-dimensional space structure of the enzyme protein, and exposing an active group; (2) blocking the exposed active group, wherein the blocking is irreversible blocking; (3) releasing a fluorescent substance; and (4) cleaning. For the first time, on the basis of a strategy of opening a disulfide bond of the enzyme protein and then performing irreversible blocking so as to better release the wound dyes substance, a background signal in the sequencing is reduced, and a signal-to-noise ratio and sequencing quality are improved, thereby facilitating efficient high-throughput sequencing.