Multiplex Amplification via Segmented Universal Primers
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Solution Overview
Problem
Current multiplex PCR methods face challenges in efficiently amplifying multiple nucleic acid sequences simultaneously due to the formation of primer-dimer interactions and spurious amplification products, limiting their application to about 10-20 reactions before undesired products predominate, and existing methods for improving these are not entirely effective.
Innovation Solution
The method involves attaching common sequences to specific nucleic acid targets for amplification using a series of oligonucleotide hybridization, extension, and ligation steps, allowing for multiplex reactions with common primers and reducing spurious product formation through the use of cleavable nucleotides and biotinylated markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple pairs of target-specific primers are combined in the same tube for multiplex PCR, then the number of simultaneous amplification reactions increases, but the frequency of formation of primer-dimer and spurious amplification products increases exponentially
Solution Approach 1:
The method divides the amplification process into two distinct rounds: a first round using target-specific primers to generate initial amplicons, and a second round using universal primers to amplify all targets simultaneously. This segmentation prevents primer-dimer formation by separating specific primer binding from universal amplification, allowing multiplexing of many more targets without exponential increase in spurious products
Solution Approach 2:
The patent introduces universal primer sequences as intermediaries that bind to common regions on all amplicons generated in the first round. These universal primers serve as mediators that enable simultaneous amplification of multiple targets without requiring multiple pairs of specific primers to coexist in the same reaction, thereby eliminating primer-dimer interactions while maintaining high productivity
2Reliability
If conventional multiplex PCR is limited to about 10-20 simultaneous amplification reactions to avoid spurious products, then primer-dimer formation is reduced, but the scalability and cost-effectiveness of large scale projects is compromised
Solution Approach 1:
By segmenting the PCR process into two rounds with different primer types, the method enables scaling to hundreds of simultaneous amplification reactions while maintaining specificity. The first round generates target-specific amplicons with high specificity, and the second round uses universal primers to amplify all targets without primer-dimer formation, achieving both reliability and high productivity
Solution Approach 2:
The universal primer sequences serve multiple functions: they bind to common regions on all amplicons regardless of target identity, enable simultaneous amplification of numerous targets, and eliminate the need for multiple specific primer pairs. This universality allows the system to scale to hundreds of reactions while maintaining amplification specificity through the standardized universal binding mechanism
3Object-generated harmful factors
If existing multiplex methods use two rounds of amplification with specific and universal primers, then spurious product formation is reduced, but the complexity of the amplification protocol increases
Solution Approach 1:
The method merges the first and second amplification rounds into a single combined reaction mixture containing both target-specific and universal primers. The reaction automatically progresses through both rounds sequentially, eliminating the need for separate reaction setups, multiple centrifugation steps, and separate thermal cycling programs, thereby reducing protocol complexity while maintaining the benefit of reduced spurious products
Solution Approach 2:
The target-specific primers perform preliminary amplification of individual targets in the first round, generating amplicons with universal binding sites. This preliminary action prepares the substrate for the second round of universal amplification, allowing both rounds to occur efficiently in a single combined reaction without requiring separate setup steps, thus reducing overall protocol complexity
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 efficient and cost-effective simultaneous amplification of multiple nucleic acid sequences, reducing spurious products and allowing for the analysis of small RNAs and degraded RNA samples by converting them into amplifiable barcode sequences, thereby enhancing the specificity and efficiency of PCR reactions.
Implementation Method 1
a first oligonucleotide, containing the priming sequence at or near the 5' end, is hybridized to the target sequence
Implementation Method 2
extended by DNA synthesis using the target as template
Implementation Method 3
The first oligonucleotide sequence is attached to the 5' end of the newly synthesized sequence of interest
Data Source
AI summary
Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.