Nuclease-Resistant Sequencing Primer for Single-Step PCR Cleanup
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Solution Overview
Problem
Standard PCR/sequencing workflows require additional reagent addition steps for cleanup, increasing labor, error risk, and automation challenges due to excess amplification primers and dNTPs, leading to aberrant sequence ladders and weak sequencing signals.
Innovation Solution
A method utilizing nuclease-resistant sequencing primers that degrade excess amplification primers, allowing for a single-step PCR/sequencing process without a separate cleanup step, using a nuclease-resistant sequencing primer that is not susceptible to exonuclease cleavage, enabling direct addition of sequencing reagents to PCR products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a standard PCR/sequencing workflow is used with cleanup steps, then sequencing accuracy is maintained, but the process complexity and labor requirements increase
Solution Approach 1:
The patent combines the PCR amplification and sequencing reactions into a single unified workflow by using amplification primers with different chemical properties (nuclease-sensitive vs nuclease-resistant). The nuclease-sensitive amplification primers are degraded during the sequencing reaction, while the nuclease-resistant sequencing primers remain intact, allowing both reactions to occur in the same tube without requiring separate cleanup steps.
Solution Approach 2:
The patent applies different chemical modifications to different primers within the same reaction system. Amplification primers are designed to be nuclease-sensitive while sequencing primers are made nuclease-resistant through chemical modification. This local differentiation in chemical properties allows the system to automatically distinguish between amplification and sequencing functions without additional processing steps.
2Productivity
If excess amplification primers are not removed before sequencing, then the workflow is simplified, but aberrant sequence ladders and sequencing errors occur
Solution Approach 1:
The patent selectively removes excess amplification primers from the reaction mixture by exploiting their nuclease sensitivity. During the sequencing reaction, nucleases degrade the nuclease-sensitive amplification primers while leaving the nuclease-resistant sequencing primers and amplification products intact, effectively extracting the harmful excess primers in situ.
Solution Approach 2:
The patent converts the potential harm of excess amplification primers into a beneficial selection mechanism. By making amplification primers nuclease-sensitive and sequencing primers nuclease-resistant, the system uses nuclease activity to selectively eliminate amplification primers while preserving sequencing primers, turning what would be interference into a useful purification step.
3Manufacturing precision
If multiple reagent addition steps are required, then reagent purity is maintained, but the time required for sample preparation increases
Solution Approach 1:
The patent merges multiple separate reagent addition steps (PCR reagents and sequencing reagents) into a single combined reaction mixture. By designing primers with differential nuclease sensitivity, the system maintains functional separation of amplification and sequencing reagents while physically combining them in one tube, eliminating sequential addition steps and reducing preparation time.
4Reliability
If nuclease treatment is applied to remove excess primers, then sequencing quality improves, but the risk of degrading sequencing reagents increases
Solution Approach 1:
The patent applies nuclease resistance selectively to sequencing primers through chemical modification, while leaving amplification primers nuclease-sensitive. This local quality differentiation protects sequencing reagents from nuclease degradation while allowing excess amplification primers to be removed, eliminating the risk of sequencing reagent damage.
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 simplifies the workflow, reduces processing steps, and generates clean, accurate sequencing data by eliminating the need for extra reagent additions, thereby reducing errors and improving automation feasibility.
Implementation Method 1
contacting the amplification reaction products with a reaction mixture comprising a nuclease and a nuclease-resistant sequencing primer, under conditions in which the excess amplification primer is degraded by the nuclease
Implementation Method 2
The hydrolytic properties of exonuclease I degrade single-stranded DNA present in the PCR mixture allowing the amplification product (amplicon) to be used more efficiently in subsequent sequencing applications
Implementation Method 3
The enzyme activity of alkaline phosphatase dephosphorylates dNTPs remaining from the PCR reaction
Data Source
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AI summary
A composition for sequencing DNA is provided and comprises a nuclease and a nuclease-resistant sequencing primer. A method of preparing DNA for sequencing and a method of sequencing DNA are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition under conditions in which excess amplification primer is degraded by the nuclease and the nuclease-resistant sequencing primer is essentially non-degraded.