Nucleotide Analog Primers to Reduce Dimers in Multiplex PCR
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Solution Overview
Problem
Existing oligonucleotide primers often form undesirable primer dimers and off-target hybrids, limiting the number of targets that can be amplified in PCR and other nucleic acid amplification methods, especially in multiplexed reactions.
Innovation Solution
Development of nucleotide analogs, such as 3-(alkynyl)-5-(1′-beta-D-2′-deoxyribofuranosyl)-pyrid-2-one (t) for thymidine, 2-aminopurine (a) for adenine, inosine (g) for guanine, and N-ethylcytidine (c) for cytidine, which reduce primer-primer interactions and maintain duplex stability, allowing for self-avoidance and efficient amplification of multiple targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard oligonucleotide primers are used for amplification, then amplification efficiency is maintained, but primer dimers and off-target hybrids form uncontrollably
Solution Approach 1:
The patent applies local quality by modifying specific nucleotide positions within primer sequences with self-avoiding analogs (G*, A*, C*, T*) while leaving other positions as standard nucleotides. This localized modification at strategic positions prevents primer-primer interactions while preserving target binding capability, thereby maintaining amplification efficiency while eliminating primer dimer formation.
Solution Approach 2:
The patent changes the chemical parameters of nucleotide building blocks by replacing standard nucleotides with self-avoiding analogs that have modified base-pairing properties. These parameter changes in nucleotide composition fundamentally alter primer interaction characteristics, preventing unwanted primer-dimer formation while maintaining productive target amplification.
2Adaptability or versatility
If the number of primer oligonucleotides is increased for multiplexed PCR, then more targets can be amplified, but primer-primer interactions increase approximately with the square of the number of targets
Solution Approach 1:
The patent applies universality by developing a general class of self-avoiding nucleotide analogs (G*, A*, C*, T*) that can be incorporated into any primer sequence regardless of target identity. This universal approach allows simultaneous use of multiple primers for multiplexed amplification of many targets while each primer maintains self-avoidance properties, preventing the quadratic increase in primer-primer interactions.
Solution Approach 2:
The patent changes the fundamental interaction parameters of primers by incorporating self-avoiding nucleotide analogs, which alter the binding characteristics to prevent off-target interactions. This parameter change enables scalable multiplexing where the number of amplifiable targets can increase without proportionally increasing harmful primer-primer interactions.
3Object-generated harmful factors
If self-avoiding oligonucleotides with modified nucleotides are used, then primer dimer formation is reduced, but hybridization stability to target may be compromised
Solution Approach 1:
The patent applies local quality by strategically placing self-avoiding nucleotide analogs at specific positions within the primer sequence where they prevent primer-dimer formation while minimizing impact on target hybridization stability. The selective positioning ensures that self-avoidance functionality is achieved without compromising the overall duplex stability needed for effective amplification.
Solution Approach 2:
The patent uses composite materials by combining self-avoiding nucleotide analogs with standard nucleotides in specific ratios and positions within the primer sequence. This composite approach balances the self-avoidance property (reducing primer dimers) with the hybridization stability property (maintaining target binding), achieving both objectives simultaneously.
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 nucleotide analogs effectively prevent primer dimers and off-target amplification, enabling successful amplification of up to 20 or more targets in multiplexed PCR and other nucleic acid amplification methods, with improved efficiency and specificity.
Implementation Method 1
hybridize, or hybridization, at a preselected temperature in water having preselected co-solutes (e.g. buffers or salts)... hybridization of canonical oligonucleotide primers to target oligonucleotides following well-known WCF base pairs
Implementation Method 2
one can serve as a template for polymerase-catalyzed extension of the other
Data Source
AI summary
This invention covers processes that amplify multiple DNA and RNA targets, solving the “multiplexed PCR problem”, as well as supporting isothermal amplification and delivering other capabilities in multiplexed molecular analysis. It uses inventive DNA analogs.