Fluorescent PCR Primer-Activated Polymerization Specificity
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Solution Overview
Problem
Conventional PCR methods are not suitable for detecting trace amounts of mutant sequences in the presence of large amounts of wild-type DNA due to non-specific amplification, and existing fluorescent detection methods suffer from poor specificity and false positives, especially when using pyrophosphorolysis-activated polymerization (PAP) with DNA polymerases lacking 5′→3′ exonuclease activity.
Innovation Solution
A method combining primer-activated polymerization with specific fluorescence-labeled probes, using a nucleic acid polymerase with 5′→3′ polymerase and exonuclease activity, and a deblocking agent to activate blocked primers, allowing for precise detection of target sequences through fluorescence signaling, while avoiding non-specific amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR reactions are used to amplify DNA fragments, then amplification efficiency is improved, but specificity deteriorates due to non-specific amplification in the presence of large amounts of non-target sequence DNA
Solution Approach 1:
The primer is segmented into two functional parts: a 5' portion that binds to the template DNA and a 3' blocked portion that prevents extension. This segmentation allows the primer to bind specifically to target sequences while the block prevents non-specific amplification, resolving the contradiction between amplification efficiency and detection specificity.
Solution Approach 2:
The primer is pre-modified with a blocking group at the 3' end before the reaction. This preliminary action ensures that the primer can bind to the template but cannot initiate extension unless the block is removed, thereby preventing non-specific amplification while maintaining the ability to amplify target sequences efficiently when conditions are correct.
2Measurement precision
If blocked primers are used in primer-activated polymerization to improve specificity, then false positives are reduced, but detection sensitivity deteriorates due to the additional activation step required
Solution Approach 1:
A blocking group serves as an intermediary element that temporarily prevents primer extension. This intermediary control mechanism ensures that extension only occurs when the blocking group is removed under specific conditions, thereby maintaining high specificity while allowing sensitive detection of target sequences when the activation conditions are met.
3Ease of manufacture
If DNA polymerases without 5'→3' exonuclease activity are used in PAP reactions, then primer activation is achieved, but probe hydrolysis cannot occur leading to poor fluorescent detection specificity
Solution Approach 1:
The patent merges two previously separate functions into a single DNA polymerase enzyme: the ability to perform pyrophosphorolysis (to activate blocked primers) and the ability to perform 5'→3' exonuclease activity (to hydrolyze probes). This combined enzyme system resolves the contradiction by enabling both primer activation and probe hydrolysis, thereby achieving both ease of manufacture and high fluorescent detection specificity.
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 highly selective and specific detection of nucleic acids, reducing false positives and allowing for multiplex detection of multiple targets with high sensitivity and specificity, overcoming the limitations of conventional PCR and PAP methods.
Implementation Method 1
wherein the fluorophore will fluoresce once the 3′ terminal blocker of the primer is removed by pyrophosphorolysis
Implementation Method 2
a quencher attached to a blocked nucleotide at the 3′ end. The fluorophore will fluoresce once the 3′ terminal blocker of the primer is removed by pyrophosphorolysis
Implementation Method 3
utilizes the pyrophosphorolysis serially coupled with polymerization by DNA polymerase for nucleic acid amplification
Data Source
AI summary
The present invention relates to a method for detecting nucleic acids by fluorescent PCR. The method combines primer-activated polymerization reaction and specific fluorescence-labeled probe, which detects the target nucleic acid with high selectivity and high specificity.