PCR Additive Oligonucleotides for Mis-Priming Suppression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing PCR reagents fail to effectively suppress mis-priming and improve polymerase selectivity without significant concentration-dependent inhibition of enzyme activities.
Innovation Solution
The use of a new class of reagents comprising two complementary oligonucleotides with specific labeling and concentration ratios, including a bulky non-planar fluorophore and a quencher, enhances polymerase selectivity by interacting with the DNA polymerase's 5′ exonuclease site, thereby improving primer specificity and reducing mis-priming.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing PCR reagents are used to improve primer specificity, then polymerase selectivity is enhanced, but enzyme activity is significantly inhibited in a concentration-dependent manner
Solution Approach 1:
The patent changes the chemical parameters of the reagent by using planar quencher moieties instead of bulky non-planar groups, and by optimizing the concentration range to 1-100 nM. This parameter change allows the reagent to enhance polymerase selectivity while avoiding significant inhibition of enzyme activity, resolving the contradiction between reliability and productivity
Solution Approach 2:
The patent creates a composite reagent structure combining specific oligonucleotide sequences with planar quencher moieties (such as dabcyl) attached at defined positions. This composite material achieves both improved polymerase selectivity and maintained enzyme activity, unlike previous single-component reagents that caused concentration-dependent inhibition
2Illumination intensity
If bulky non-planar fluorophores are used as modifying groups, then fluorescence signal is enhanced, but polymerase selectivity is reduced
Solution Approach 1:
The patent applies local quality by placing planar quencher moieties at specific local positions (5′ or 3′ ends) of the oligonucleotide rather than using bulky groups throughout. This localized modification with planar structures maintains polymerase selectivity while still providing fluorescence quenching functionality when paired with fluorophores
Solution Approach 2:
The patent uses planar quencher moieties that can be easily synthesized and attached to oligonucleotides, replacing the need for bulky non-planar groups. This copying approach with planar structures achieves the same quenching function without the detrimental effect on polymerase selectivity
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
These reagents enhance polymerase selectivity by suppressing mis-priming and maintaining enzyme activity, leading to improved PCR accuracy and reduced scatter among replicate reactions.
Implementation Method 1
pairs of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long
Implementation Method 2
complementary nucleotides of the nine-nucleotide-long stem
Implementation Method 3
oligonucleotides are modified on one or both ends by addition of polycyclic moieties, for example, dabcyl moieties, that do not have bulky portions that are non-planar. Bulky non-planar groups, such as fluorescein (FAM) were tested and judged to be not useful as modifying groups in PCR additives
Data Source
AI summary
Provided herein are reagents for improving PCR accuracy.