Universal Base Primers for Multiplex PCR Dimer Suppression
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Solution Overview
Problem
Existing methods for multiplex nucleic acid amplification face challenges in reducing primer dimer formation, which leads to inefficient amplification and waste of reagents, particularly in reactions involving multiple target nucleic acid molecules.
Innovation Solution
Incorporating universal base nucleotides into primer pairs, specifically in the core region of the 3′-end, to inhibit the formation of both-strands extendable primer dimers, thereby enhancing amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional primers are used in multiplex amplification reactions, then amplification of target nucleic acid molecules can proceed, but primer dimer formation occurs which reduces amplification efficiency
Solution Approach 1:
The patent introduces universal base nucleotides at specific positions (3rd to 6th nucleotide from the 3'-end) within the primer sequence. This local modification creates different binding properties at the primer's core region while maintaining overall primer functionality, effectively preventing primer dimer formation without compromising target amplification efficiency
Solution Approach 2:
The patent changes the chemical composition parameters of the primer by incorporating universal base nucleotides (such as inosine or 7-deaza-2'-deoxyinosine) that can pair with multiple standard nucleotides. This parameter change in base composition allows the primer to maintain flexibility in binding to target sequences while reducing non-specific binding to other primers, thereby eliminating primer dimer formation
2Object-generated harmful factors
If primer sequence or length is altered to minimize primer dimer formation, then primer dimer formation is reduced, but it becomes difficult to apply to multiplex amplification reactions
Solution Approach 1:
The patent employs universal base nucleotides that can pair with multiple standard nucleotides (A, T, C, G), giving the primer multi-functional binding capability. This universality allows the same primer design strategy to be applied across multiple primer pairs in multiplex amplification reactions, maintaining adaptability while preventing primer dimer formation through the inherent properties of universal bases
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 effectively reduces primer dimer formation, improving the efficiency of multiplex amplification reactions by minimizing the formation of both-strands extendable primer dimers and optimizing the use of reagents.
Implementation Method 1
each primer comprises a hybridizing nucleotide sequence to a corresponding target nucleic acid molecule
Implementation Method 2
the UBP has 1-3 universal base nucleotides; wherein one or two of the universal base nucleotides are located in a core region ranging from 3rd nucleotide to 6th nucleotide at the 3′-end of the UBP
Implementation Method 3
performing a multiplex amplification reaction comprising at least two cycles of primer annealing, primer extension and denaturation
Implementation Method 4
repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template
Data Source
AI summary
The present invention relates to a method for amplifying at least three target nucleic acid molecules with reduced primer dimer formation in a multiplex amplification reaction. The method of present invention can inhibit primer dimer formation and hence generation of nonspecific amplification products in an effective manner in a multiplex amplification reaction for at least three target nucleic acid molecules.