Single-Tube PCR Method for Amplicon Construct Generation
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Solution Overview
Problem
Conventional PCR methods for generating amplicon constructs are cumbersome, expensive, and prone to cross-contamination, especially when dealing with complex constructs required for next-generation sequencing, due to the need for multiple steps and open tube transfers.
Innovation Solution
A single-tube PCR method that uses an oligonucleotide probe with a universal sequence and a universal primer to generate target-specific primers in situ, allowing for the amplification of complex amplicon constructs with reduced risk of contamination and increased specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional two-step PCR method is used to generate complex amplicon constructs, then functional sequences can be added to amplicons, but the process becomes cumbersome and time-consuming due to multiple steps and open tube transfers
Solution Approach 1:
The patent combines the primary and secondary PCR steps into a single reaction mixture, eliminating the need for separate tube transfers and multiple processing steps. The universal primers and gene-specific primers coexist in one tube, with the universal primers amplifying the universal sequences first, then serving as templates for the gene-specific primers to add functional sequences, all within a single PCR reaction.
Solution Approach 2:
The universal sequences are pre-added to the gene-specific primers before the PCR reaction begins. This preliminary incorporation of functional sequences into the primer design allows the amplification process to simultaneously create the amplicon and attach the required functional elements, eliminating post-PCR modification steps.
2Reliability
If conventional two-step PCR method with open tube transfers is used, then amplicons can be generated, but cross-contamination and carry-over risks increase significantly
Solution Approach 1:
By merging both PCR steps into a single closed tube reaction, the patent eliminates all open tube transfers between steps. The entire amplification process, including universal sequence amplification and functional sequence addition, occurs within one sealed reaction vessel, completely preventing cross-contamination and carry-over errors.
3Ease of operation
If conventional one-step PCR method is used to add functional sequences, then the process is simple, but it becomes expensive and cumbersome when large numbers of different functional sequences are required
Solution Approach 1:
The patent creates a universal system where a single set of universal primers can work with multiple different gene-specific primers, each containing different functional sequences. The universal sequences act as a common platform that accepts various functional elements, allowing one PCR reaction setup to generate numerous different amplicon constructs with diverse functional sequences.
Solution Approach 2:
The primer design is segmented into modular components: a universal sequence portion that provides the common amplification target, and a gene-specific portion that contains the functional sequence to be added. This segmentation allows the functional sequences to be independently designed and combined with the universal primers, simplifying the overall process when multiple functional sequences are needed.
4Adaptability or versatility
If secondary PCR step with universal primers is used, then complex combinations of functional sequences can be added, but the risk of amplifying incorrect amplicons increases
Solution Approach 1:
By combining both PCR steps in one reaction, the patent ensures that only amplicons that successfully undergo both the universal sequence amplification and the functional sequence addition in the same reaction tube are produced. This eliminates the risk of universal primers amplifying incorrect targets from previous reactions, as there are no tube transfers or separate reaction wells involved.
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 simplifies the process, reduces costs, enhances targeting specificity, and minimizes the risk of amplifying incorrect targets, while enabling the generation of complex amplicon constructs suitable for sequencing applications like NGS.
Implementation Method 1
The primers used in the secondary PCR step are designed such that their 3′ ends are complimentary to and hybridise to the universal sequences
Implementation Method 2
extension of the primers by a DNA polymerase
Implementation Method 3
denaturation of the DNA template
Data Source
AI summary
A method for generating amplicon constructs of a target sequence is disclosed, the method comprising providing a target sequence; an oligonucleotide probe, comprising a universal sequence and further comprising, at or towards its 5′ end, a target specific sequence capable of hybridising to the reverse complement of a sequence at, or flanking one of the 3′ ends of the target sequence; a universal primer, comprising at its 3′ end a sequence capable of hybridising to the universal sequence of the oligonucleotide probe and performing a Polymerase Chain Reaction (PCR).