Enhanced Solid Phase PCR for Single-Stranded DNA Generation
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Solution Overview
Problem
Existing methods for generating single-stranded nucleic acid molecules, such as PCR, are inefficient due to kinetic constraints and require excessive handling, leading to contamination risks and increased processing time and cost, especially in diagnostic contexts.
Innovation Solution
The method employs primers with differential priming properties at specific annealing conditions, allowing for efficient generation of single-stranded DNA molecules by altering annealing conditions during the amplification process, known as Enhanced Solid Phase-PCR (ESP-PCR), which enhances solid phase amplification without the need for additional sample handling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If asymmetric PCR is used to generate ssDNA by employing an imbalanced primer pair concentration, then single-stranded DNA production is achieved, but the process is inherently limited in processivity due to the limiting concentration of one primer
Solution Approach 1:
The patent divides the amplification process into two distinct phases: exponential amplification phase using both primers to generate dsDNA, and linear amplification phase using only the excess primer to generate ssDNA. This segmentation allows each phase to optimize for its specific purpose, resolving the contradiction between ssDNA production and processivity.
Solution Approach 2:
The patent performs exponential amplification first to generate sufficient dsDNA templates before initiating the linear amplification phase. This preliminary action ensures that when ssDNA production begins, there is abundant template material available, thereby improving processivity without compromising ssDNA yield.
2Quantity of substance
If competitor primer asymmetric PCR is employed by separate addition of competitor primer following PCR thermocycling, then ssDNA generation is achieved, but excessive handling is required which increases contamination risk, processing time and cost
Solution Approach 1:
The patent combines the exponential and linear amplification phases into a single continuous reaction by including both primers in the initial reaction mixture. The transition from exponential to linear phase occurs automatically during thermocycling as the excess primer becomes limiting, eliminating the need for separate primer addition steps and reducing handling requirements.
Solution Approach 2:
The patent maintains continuous amplification action throughout the thermocycling process without interrupting to add or remove components. The reaction seamlessly transitions from exponential to linear phase, ensuring continuous ssDNA generation while minimizing handling steps and contamination risk.
3Quantity of substance
If solid phase matrices are labeled with PCR products using symmetric or asymmetric PCR, then solid support labeling is achieved, but the approaches are relatively inefficient due to kinetic constraints and low effective substrate concentrations
Solution Approach 1:
The patent performs preliminary exponential amplification to generate high concentrations of dsDNA templates before the linear amplification phase. This ensures that when the reaction occurs on solid phase matrices, there is sufficient substrate concentration to overcome kinetic constraints and achieve efficient labeling.
Solution Approach 2:
The patent changes the effective concentration parameter dynamically during the reaction by transitioning from a regime with high primer concentration (exponential phase) to low primer concentration (linear phase). This parameter change optimizes the reaction kinetics for different stages of amplification, improving overall labeling efficiency on solid supports.
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
ESP-PCR achieves high loading of amplicon-associated signals on solid supports with improved sensitivity and reduced amplification bias, enabling uncompromised and sensitive solid phase amplification without additional processing steps.
Implementation Method 1
After amplifying target sequences using the polymerase chain reaction (PCR)
Implementation Method 2
Following a period of exponential amplification in the presence of permissive annealing conditions
Implementation Method 3
enhanced solid phase polynucleotide amplification
Data Source
AI summary
The present invention relates generally to methods for generating single stranded nucleic acid molecules following enhanced solid phase polynucleotide amplification. The present invention employs an amplification reaction using primers with differential priming properties at particular annealing conditions or an immobilized primer nested between two aqueous phase primers. Thus, by primer design, solid support primer participation is enhanced relative to aqueous phase primers. The subject invention further provides methods for labeling solid matrices with single and double stranded nucleic acid molecules. Kits for generating single stranded nucleic acid molecules and for conducting amplification reactions also form part of the present invention. The present invention further provides amplification systems for the generation of single stranded nucleic acid molecules optionally labelled with a reporter molecule and their use inter alia as labels, primers and probes.