Controlled Strand-Displacement for Synchronized Paired-End Sequencing
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Solution Overview
Problem
Existing paired-end sequencing methods struggle to maximize the number of second strands with optimized lengths for sequencing and synchronize their production efficiently, leading to inefficiencies in read alignment and mutation detection.
Innovation Solution
The method involves controlled production of complementary DNA strands using multiple displacement amplification (MDA) with synchronized strand production, utilizing excisable nucleotides and reversible blocking groups to generate multiple second strands, and optimizing polymerase concentration and conditions for synchronized extension.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple displacement amplification (MDA) is performed to generate second strands, then the quantity of second strands is increased, but the synchronization and length optimization of strand production deteriorates
Solution Approach 1:
The patent applies preliminary action by first generating first read strands from the DNA template before performing MDA to generate second strands. This sequential approach ensures that second strand production is coordinated with the sequencing workflow, improving synchronization. The method also includes optional steps of removing excess polymerase and controlling reaction conditions to optimize second strand length for subsequent sequencing operations.
Solution Approach 2:
The patent employs dynamics by controlling the MDA reaction conditions, including polymerase concentration, temperature, and reaction time, to dynamically adjust the production rate and length of second strands. The method allows for removal of excess polymerase during the reaction to prevent over-extension, thereby optimizing strand length distribution and synchronization with the sequencing process.
2Productivity
If polymerase concentration is increased to maximize second strand production, then productivity is improved, but manufacturing precision of strand length deteriorates
Solution Approach 1:
The patent applies periodic action by performing MDA in controlled intervals with optional removal of excess polymerase during the reaction. This periodic control prevents continuous uncontrolled extension, thereby maintaining strand length uniformity while still achieving high productivity through optimized reaction conditions and timing.
Solution Approach 2:
The patent uses parameter changes by optimizing polymerase concentration, temperature, and reaction time to achieve both high productivity and precise strand length control. The method includes adjusting these parameters during the MDA process to maximize second strand yield while maintaining uniform lengths suitable for sequencing.
3Manufacturing precision
If controlled MDA is performed to synchronize second strand production, then manufacturing precision is improved, but productivity may be reduced due to controlled conditions
Solution Approach 1:
The patent applies preliminary action by performing first read sequencing before MDA-based second strand synthesis. This preliminary step establishes a synchronized workflow where second strands are produced after and coordinated with the first read process, ensuring manufacturing precision while maintaining overall productivity through efficient workflow design.
Solution Approach 2:
The patent maintains continuity of useful action by performing MDA under controlled conditions that allow second strand synthesis to proceed continuously and synchronously with the sequencing workflow. The controlled environment ensures continuous production of properly lengthed second strands without interruption, balancing precision and productivity.
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 enhances the yield and synchronization of second strands, improving read alignment and mutation detection efficiency in paired-end sequencing.
Implementation Method 1
performing controlled multiple displacement amplification (MDA) by extending the first read strands or portions thereof with a polymerase having strand-displacement activity to generate a plurality of second strands
Implementation Method 2
extending first read primers hybridized on a plurality of single strand DNA concatemers immobilized on an array to generate first reads
Data Source
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AI summary
This application relates to methods and compositions used for paired-end sequencing. The method comprises providing a DNA array comprising a surface immobilized with DNA concatemers. For each of the plurality of DNA concatemers on the array, the method comprises annealing first read primers to primer binding sites on the DNA concatemer, extending at least some of the first read primers to incorporate dNTPs or dNTP analogs, thereby producing first read strands, wherein each of the dNTPs or dNTP analogs being incorporated is identified to produce first reads, performing controlled MDA by extending at least some of the first read strands with a polymerase having strand-displacement activity to generate a plurality of second strands, each second strand comprising a portion that is hybridized to the DNA concatemer, and an unhybridized, single-stranded branch; and annealing second read primers to the single-stranded branches of the plurality of second strands, and extending the second read primers to generate the second reads.