Multiplex Amplification via Flap Nuclease Circularization
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Solution Overview
Problem
Traditional multiplex PCR methods face challenges with uneven or failed amplification of GC-rich sequences, preferential amplification, poor sensitivity and specificity, and the generation of spurious products, limiting their ability to amplify multiple nucleic acid sequences uniformly and efficiently.
Innovation Solution
The method involves fragmenting targets, circularizing them using splinting circularization probes, and ligating the ends, followed by rolling circle amplification, which allows for the specific and uniform amplification of multiple sequences without generating spurious products, using techniques such as 5' and 3' flap formation and removal by specific nucleases, and subsequent ligation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional multiplex PCR is used to amplify multiple nucleic acid sequences, then amplification of multiple targets is achieved, but uneven or failed amplification of GC-rich sequences occurs and spurious products are generated
Solution Approach 1:
The patent divides the amplification process into two distinct stages: (1) target-specific primer extension to generate flap structures, and (2) flap nuclease processing followed by common oligo ligation and rolling circle amplification. This segmentation allows each stage to be optimized independently, resolving the contradiction between multiplex capability and amplification reliability.
Solution Approach 2:
The patent introduces flap nucleases as intermediary enzymes that process the flap structures generated by target-specific primers. These nucleases act as mediators between the specific primer extension step and the common oligo ligation step, enabling uniform processing of diverse GC-rich sequences and eliminating spurious products while maintaining multiplex amplification capability.
2Reliability
If primer concentrations and reaction conditions are adjusted to improve amplification uniformity, then some amplification problems are reduced, but the complexity of optimization increases
Solution Approach 1:
The patent changes the fundamental reaction parameters by introducing flap nucleases and rolling circle amplification instead of traditional PCR cycling. This parameter change shifts the optimization focus from complex primer concentration balancing to enzyme activity optimization, reducing the overall optimization complexity while improving amplification uniformity.
Solution Approach 2:
The patent extracts the problematic PCR amplification step and replaces it with rolling circle amplification of ligated products. This extraction removes the source of uneven amplification and spurious product generation, simplifying the optimization requirements while maintaining reliability.
3Reliability
If subcycling temperatures are used during annealing and elongation, then amplification of some templates is improved, but the amplification of other templates suffers and spurious products are generated
Solution Approach 1:
The patent performs preliminary target-specific primer extension to generate flap structures before the main amplification step. This preliminary action allows specific binding to occur without the harmful effects of subcycling, and the subsequent flap nuclease processing ensures only correctly bound primers are amplified, eliminating spurious products.
Solution Approach 2:
The patent converts the potential harm of non-specific binding during annealing into a benefit by using flap nuclease processing. The flap structures generated by non-specific binding are removed by nucleases, while correctly bound primers generate proper flap structures that are protected and amplified. This converts the harmful non-specific amplification into a selective enrichment process.
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 enables the simultaneous amplification of over 100, 1000, or 50,000 different sequence targets in a single reaction, providing high specificity and uniformity, suitable for genotyping, copy number, methylation, translocation, or sequence analysis, and can be scaled for various applications including resequencing and genotyping.
Implementation Method 1
A 5' flap nuclease is used to remove the 5' flap and to generate juxtaposed ends for ligation
Implementation Method 2
ligating the ends, followed by rolling circle amplification
Implementation Method 3
followed by rolling circle amplification, which allows for the specific and uniform amplification of multiple sequences
Data Source
AI summary
Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5′ or 3′ flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5′ and 3′ flaps are generated. The flaps are cleaved using 5′ or 3′ flap endonucleases or 3′ to 5′ exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.