Enzyme Composition for Monoclonal Cluster Generation
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Solution Overview
Problem
Next-generation sequencing (NGS) technologies require multiple steps for producing monoclonal populations of amplicons, which are time-consuming due to incompatibilities between enzyme activities, such as exonuclease and glycosylase, that inhibit each other's performance.
Innovation Solution
Combining exonuclease and glycosylase enzymes into a single step for producing monoclonal amplicons, utilizing a composition that includes uracil DNA glycosylase, an endonuclease, and an exonuclease with 3' to 5' single-stranded DNA exonuclease activity, to create abasic sites and reduce the number of reagents needed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exonuclease and glycosylase are used in separate steps, then enzyme activity compatibility is maintained, but sequencing time increases
Solution Approach 1:
The patent combines exonuclease and glycosylase into a single enzyme composition that functions simultaneously. The enzyme mixture includes exonuclease, glycosylase, and phosphatase activities in one formulation, allowing all three enzymatic functions to occur in a single step during library preparation, thereby reducing sequencing time while maintaining enzyme compatibility through carefully balanced formulation.
Solution Approach 2:
The enzyme composition is designed to perform multiple functions simultaneously - exonuclease activity for 3' exonuclease digestion, glycosylase activity for base removal, and phosphatase activity for phosphate removal. This multi-functional enzyme system eliminates the need for separate treatment steps while maintaining the reliability of each individual enzymatic function.
2Reliability
If multiple separate steps are used for producing monoclonal amplicons, then process compatibility is maintained, but the number of reagents increases
Solution Approach 1:
The patent merges multiple enzyme treatments into a single composition containing exonuclease, glycosylase, and phosphatase. This consolidated enzyme mixture replaces what would traditionally require separate application steps with different reagents, reducing the total number of reagents needed while maintaining process compatibility through optimized enzyme formulation.
Solution Approach 2:
The enzyme composition serves as a universal reagent that performs multiple enzymatic functions simultaneously - exonuclease digestion, glycosylase base removal, and phosphatase phosphate removal. This single multi-functional reagent replaces multiple separate reagents, reducing complexity and reagent quantity while maintaining process reliability.
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 combination reduces the sequencing time, maintains primary metrics and read quality, and decreases costs by performing two steps simultaneously without detrimental effects on data output or genome build metrics.
Implementation Method 1
uracil DNA glycosylase... to produce an abasic site at the cleavage site
Implementation Method 2
exonuclease having a 3′ to 5′ single-stranded DNA exonuclease activity
Data Source
AI summary
The present disclosure is concerned with compositions and methods for reducing the steps used in the generation of monoclonal clusters by combining the enzymes used for linearization and removal of unused surface primers.