Multiplex PCR Primer Sets for Uniform Amplification

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Solution Overview

Problem

Multiplex PCR faces challenges such as mispriming, formation of unwanted side products, and uneven amplification due to nonspecific primer binding and cross-hybridization, which complicates the amplification of multiple DNA sequences simultaneously.

Innovation Solution

Sets of experimentally validated gene-specific primer pairs, including 10 or more pairs with forward and reverse primers, are provided, some of which incorporate an anchor domain for universal primer binding, designed to optimize conditions for high-throughput sequencing applications.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If multiple primer pairs are used in multiplex PCR to amplify many DNA sequences simultaneously, then productivity increases, but mispriming and formation of unwanted side products occur due to nonspecific primer binding

Engineering Contradiction:

Improveamplification of multiple DNA sequencesVSAvoidspecificity of primer binding

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by optimizing primer design parameters including length (18-25 nucleotides), GC content (40-60%), melting temperature (55-65°C), and secondary structure-free sequences. These parameter optimizations ensure specific binding while enabling multiplex amplification of multiple DNA sequences simultaneously without mispriming or side product formation

Inventive Principle:

Principle #35Parameter changes

2Productivity

If multiple primer pairs are used in multiplex PCR to amplify many DNA sequences simultaneously, then productivity increases, but uneven or no amplification of some target sequences occurs due to cross hybridization

Engineering Contradiction:

Improveamplification of multiple DNA sequencesVSAvoiduniformity of amplification

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent optimizes primer parameters including melting temperature uniformity (55-65°C range), GC content (40-60%), and length (18-25 nucleotides) to ensure even amplification across multiple target sequences. These parameter controls prevent cross-hybridization and achieve uniform amplification productivity across all targets in the multiplex reaction

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs iterative optimization and experimental validation of primer sets to achieve uniform amplification. Through feedback from amplification results, primer sequences are refined to eliminate cross-hybridization issues and ensure even representation of all target sequences in the final amplification product

Inventive Principle:

Principle #23Feedback

3Productivity

If primer concentration is increased to improve amplification efficiency, then productivity increases, but formation of unwanted side products such as homodimers and heterodimers increases

Engineering Contradiction:

Improveamplification efficiencyVSAvoidformation of homodimers and heterodimers

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The patent optimizes primer concentration parameters and sequence parameters to prevent dimer formation. Primers are designed with specific length (18-25 nt), GC content (40-60%), and melting temperature (55-65°C) parameters that minimize self-complementarity and cross-complementarity, enabling efficient amplification at optimal concentrations without generating homodimers or heterodimers as side products

Inventive Principle:

Principle #35Parameter changes

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 primer sets enhance the specificity and efficiency of multiplex amplification by reducing nonspecific binding and side product formation, ensuring uniform and effective amplification of target sequences in high-throughput sequencing.

Implementation Method 1

multiplex polymerase chains reactions (multiplex PCR) include the simultaneous amplification of many DNA sequences

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

mispriming due to nonsspecific primer binding to non-target templates

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 3

amplification of many DNA sequences in one reaction

Methodology Applied

Scientific EffectPolymerization:

Implementation Method 4

simultaneous amplification of many DNA sequences

Methodology Applied

Scientific EffectDNA replication:

Implementation Method 5

maintain controlled cycling and annealing temperatures

Methodology Applied

Scientific EffectDenaturation:

Implementation Method 6

maintain controlled cycling and annealing temperatures

Methodology Applied

Scientific EffectAnnealing: Annealing

Data Source

PatentUS11655510B2Experimentally validated sets of gene specific primers for use in multiplex applications

Publication Date: 2023.05.23 CELLECTA

AI summary

Sets of experimentally validated gene specific primer pairs are provided. Embodiments of the sets include 10 or more gene specific primer pairs of forward and reverse primers. The forward and reverse primers of each primer pair include gene specific primers that are experimentally validated as suitable for use in a multiplex amplification assay. In some instances, each of the forward and reverse primers includes an anchor domain that includes a universal primer binding site. The sets find use in a variety of different applications, including high-throughput sequencing applications.