Multiplex PCR Primer Pairs With Barcodes Reduce Mis-priming
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Solution Overview
Problem
Current multiplex PCR technologies face challenges in accurately amplifying and analyzing small nucleic acid samples from large numbers of biological samples due to issues like mis-priming, formation of unwanted side products, and the complexity of labeling gene-specific primers with unique barcodes, which limits their application in high-throughput sequencing and clinical diagnostics.
Innovation Solution
The development of methods to prepare sample-barcoded anchor-domain-flanked gene-specific DNA fragments using gene-specific primer pairs with barcode domains, allowing for the simultaneous amplification and identification of multiple DNA sequences, and the use of unique molecular identifiers to label each nucleic acid molecule, enabling more precise analysis of nucleic acid compositions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple primer pairs are used in multiplex PCR to amplify many DNA sequences simultaneously, then productivity is improved, but unwanted side products form due to mis-priming and inter-molecular interactions
Solution Approach 1:
The primer pool is segmented into multiple individual primer pairs, each targeting a specific DNA sequence. This segmentation allows for controlled amplification of multiple targets while reducing non-specific interactions that occur in undifferentiated primer mixtures.
Solution Approach 2:
Each primer is designed with specific local properties including optimized Tm values, GC content, and sequence composition tailored to its specific target. This local optimization reduces mis-priming and non-specific binding while maintaining efficient amplification of the intended target.
2Productivity
If primer concentrations are increased to improve amplification efficiency, then productivity is improved, but mis-priming and side product formation increase
Solution Approach 1:
The concentration of each primer in the multiplex reaction is individually optimized based on its specific amplification requirements, target abundance, and performance characteristics. This parameter optimization allows efficient amplification while minimizing non-specific binding and side product formation.
3Measurement precision
If barcodes are added to primers to label sample-specific sequences, then measurement precision is improved, but device complexity increases due to synthesis and management requirements
Solution Approach 1:
A universal barcode sequence is designed that can be attached to multiple different primers targeting different genes. This universal barcode allows sample identification across multiple targets without requiring unique barcodes for each primer, reducing synthesis complexity while maintaining precise sample tracking.
Solution Approach 2:
The barcode sequence is copied and attached to multiple different gene-specific primers. This copying approach allows a single barcode design to serve multiple primers, reducing the overall complexity of barcode management while enabling precise sample identification in multiplex assays.
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 enhances the sensitivity and specificity of multiplex PCR assays, particularly in single-cell analyses and clinical diagnostics, by reducing complexity and improving the ability to analyze hundreds or thousands of distinct nucleic acid molecules, thereby improving the quality and cost-effectiveness of biological sample analysis.
Implementation Method 1
employing a set of gene specific primer pairs, wherein each pair of gene specific primers is made up of a forward primer and a reverse primer
Implementation Method 2
Multiplex polymerase chains reactions (multiplex PCR) include the simultaneous amplification of many DNA sequences in one reaction
Data Source
AI summary
Methods of preparing a plurality of sample-barcoded anchor-domain-flanked gene specific deoxyribonucleic acid (DNA) fragments from a template nucleic acid, e.g., ribonucleic acid (RNA), sample are provided. Aspects of the methods include employing a set of gene specific primer pairs, wherein each pair of gene specific primers is made up of a forward primer and a reverse primer, at least one of which includes a sample barcode domain. The methods find use in a variety of different applications, including high-throughput sequencing, e.g., expression profiling, applications, including of small biological samples, e.g., single-cells.