Multiplexed Nucleic Acid Patch PCR Method
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Solution Overview
Problem
Current methods for sequencing a subset of the human genome are costly and inefficient, as they require separate PCR reactions for each region of interest and large amounts of DNA, limiting the ability to sequence multiple samples simultaneously and hindering the implementation of personalized medicine.
Innovation Solution
A multiplexed PCR method that amplifies multiple targeted regions from a small amount of nucleic acid using nucleic acid patch PCR, which involves creating defined ends of nucleic acid sequences, circularizing amplicons, and using universal primers for subsequent amplification, enabling the simultaneous sequencing of multiple samples with high specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate PCR reactions are performed for each region of interest, then amplification specificity is maintained, but the cost increases and throughput decreases
Solution Approach 1:
The patent combines multiple separate PCR reactions into a single multiplexed PCR reaction by using a universal primer pair that can amplify multiple different target regions simultaneously. Each target region is represented by a unique barcode sequence, allowing all regions to be amplified in one reaction mixture, thereby increasing throughput while maintaining specificity through barcode-based identification.
Solution Approach 2:
The patent employs a universal primer pair that serves multiple functions: it binds to common sequences flanking different target regions and initiates amplification for all targets simultaneously. This universal primer approach allows a single PCR reaction to perform the work of multiple separate reactions, improving productivity without sacrificing the ability to specifically amplify each target region.
2Productivity
If multiple primer pairs are used in multiplex PCR, then multiple regions can be amplified simultaneously, but mispriming events and inter-primer interactions increase
Solution Approach 1:
The patent extracts the variable element (target-specific sequence) from the primer and places it in the barcode region of the amplicon, while the functional primer elements (binding sites and universal sequences) remain constant. This separation allows multiple targets to be amplified using the same primer pair, reducing mispriming events and inter-primer interactions while maintaining the ability to specifically amplify each target through the unique barcode sequences.
Solution Approach 2:
The patent introduces a universal primer pair as an intermediary that mediates amplification of multiple different target regions. Instead of using multiple target-specific primer pairs that directly compete with each other, the universal primers act as intermediaries that bind to common flanking sequences and initiate amplification for all targets, thereby reducing direct primer-primer interactions and mispriming events.
3Quantity of substance
If a large amount of starting DNA is used to supply template for all individual PCRs, then sufficient template is available for each reaction, but DNA becomes a limiting factor for clinical samples
Solution Approach 1:
The patent merges multiple separate PCR reactions into a single multiplexed reaction, which allows the starting DNA to be distributed across multiple target amplifications in one reaction rather than requiring separate reactions for each target. This consolidation reduces the total amount of starting DNA needed while ensuring sufficient template is available for each target region through the efficient use of a single reaction mixture.
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 method allows for the efficient amplification and sequencing of multiple nucleic acid sequences from limited DNA samples, enhancing the throughput and reducing costs, thereby facilitating the analysis of genetic variations and correlations between genotype and phenotype.
Implementation Method 1
annealing an upstream primer and a downstream primer to the at least two nucleic acid sequences to be amplified
Implementation Method 2
amplifying the at least two nucleic acid sequences to create amplicons
Implementation Method 3
circularizing the amplicons by ligating an adapter nucleic acid sequence to the known 5′ and 3′ ends of the at least two linear nucleic acid sequences
Implementation Method 4
annealing an upstream nucleic acid patch and a downstream nucleic acid patch to each nucleic acid sequence of known 5′ and 3′ ends from step (a)
Data Source
AI summary
The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.