Unidel-PCR Primer Set for Insertion Deletion Mutation Detection
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Solution Overview
Problem
Current methods for detecting insertion and deletion mutations in genes are inefficient, requiring prior knowledge of mutated sequences, multiple primers for full coverage, and are prone to unspecific hybridization, leading to low sensitivity and labor-intensive processes.
Innovation Solution
A novel primer design scheme for universal insertion/deletion polymerase chain reaction (Unidel-PCR) using a blocking primer and a forward primer, where the blocking primer prevents amplification of the reference sequence, allowing a single primer set to detect insertion and deletion variants without prior knowledge of the mutated sequence, with the forward primer competing to anneal with the template strand at a different temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional primers or probes are used for detecting insertion/deletion mutations, then detection can be performed with prior knowledge of the mutated sequence, but multiple primers are required to ensure full coverage of all mutated sequences and the method becomes error-prone with lower detection sensitivity
Solution Approach 1:
The patent applies universality by designing a single primer set that can detect multiple types of insertion and deletion mutations without requiring prior knowledge of the specific mutated sequence. The primer set is engineered to universally bind to conserved regions flanking the variable insertion/deletion sites, enabling one primer set to serve multiple detection functions across different mutation types and locations.
Solution Approach 2:
The patent introduces an intermediary mechanism using a blocking primer that competes with the detection primer for binding to the template DNA. This blocking primer acts as a mediator that prevents false positive signals by selectively blocking binding at wild-type sequences while allowing detection at mutated sequences, thereby improving detection accuracy without requiring multiple separate primer sets.
2Reliability
If conventional methods are used for detecting insertion/deletion mutations, then primers can be designed with prior knowledge of the mutated site, but unspecific hybridization occurs when the mutation site is looped out during hybridization
Solution Approach 1:
The patent applies local quality by designing primers with differentiated binding characteristics - the detection primer has high affinity for mutated sequences while the blocking primer has high affinity for wild-type sequences. This local differentiation in binding specificity ensures that each primer selectively interacts with its target sequence type, preventing unspecific hybridization and improving detection reliability.
Solution Approach 2:
The patent implements preliminary anti-action by introducing a blocking primer that preemptively binds to wild-type sequences before the detection primer can bind. This blocking action prevents false positive signals from unspecific hybridization at wild-type sites, allowing the detection system to reliably distinguish mutated sequences from wild-type sequences without cross-reactivity.
3Measurement precision
If conventional methods are used for detecting insertion/deletion mutations, then detection can be performed, but higher amount of DNA and more reaction cycles are required which are labor-intensive
Solution Approach 1:
The patent applies parameter changes by optimizing the biochemical parameters of the PCR detection system - including primer concentrations, annealing temperatures, and cycle conditions - to maximize detection sensitivity. These parameter optimizations enable the detection of insertion/deletion mutations with lower amounts of DNA input and fewer reaction cycles compared to conventional methods.
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
The method achieves ultra-high sensitivity in detecting insertion/deletion variants, capable of identifying mutations even when they comprise a minor fraction of the total DNA, with reduced reaction requirements and increased accuracy.
Implementation Method 1
the hybridization of the primer or probe with the target nucleic acid might occur with a mutation site being looped out
Implementation Method 2
the forward primer competing to anneal with the template strand at a different temperature
Implementation Method 3
real-time quantitative polymerase chain reaction (RTQ-PCR) and/or apparatus for RTQ-PCR are required to accomplish the detection process
Implementation Method 4
carrying out PCR by using generic PCR primers together with a blocking primer
Data Source
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AI summary
Disclosed herein is a polymerase chain reaction (PCR)-based method for detecting an insertion/deletion variant, as compared with a reference sequence, in a selected region of a target gene in a sample. The target gene has a template strand and a coding strand complementary to the template strand. The method uses a primer set that includes a blocking primer, and a forward primer. The blocking primer and the forward primer are partially overlapping and have different melting temperatures, and the 3'-end of the blocking primer is modified to prevent the extension of the blocking primer during the PCR. Accordingly, under specific PCR conditions, the presence of the PCR product is indicative of the presence of an insertion/deletion mutation in the selected region, and the absence of the PCR product is indicative of the absence of an insertion/deletion mutation in the selected region.