Low Background Multiplex Nucleic Acid Amplification via Circularization
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Solution Overview
Problem
Conventional multiplex PCR reactions often generate significant non-target amplicons, limiting the utility of amplification products due to mis-priming events, especially when using multiple specific primer pairs, which compromises the yield and accuracy of further analysis.
Innovation Solution
The method involves contacting a nucleic acid sample with multiple primer pairs under PCR conditions, followed by circularization with target nucleic acid circularizing reagents, and subsequent selection of circularized target amplicons to reduce non-target amplicon generation, allowing for low background multiplex nucleic acid amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple specific primer pairs are added to the same PCR reaction to increase assay throughput, then the number of target nucleic acids that can be amplified simultaneously increases, but the generation of non-target amplicons increases significantly, compromising amplification yield and analysis accuracy
Solution Approach 1:
A blocking oligonucleotide is introduced as an intermediary component that specifically binds to non-target amplicons during or after PCR amplification. This blocking oligonucleotide contains a region complementary to sequences generated by mis-priming events, allowing it to selectively hybridize to and inhibit non-target amplicons while leaving target amplicons unaffected. This mediator approach enables the system to tolerate higher numbers of primer pairs by actively suppressing the harmful non-target products they generate.
Solution Approach 2:
The invention modifies the chemical parameters of the PCR reaction by incorporating modified nucleotides (such as dUTP instead of dTTP) and using blocking oligonucleotides with specific chemical modifications. These parameter changes enable selective degradation or inhibition of non-target amplicons through enzymatic treatment (e.g., uracil-DNA glycosylase), thereby changing the composition and purity of the amplification products without affecting the target sequences.
2Adaptability or versatility
If the number of primer pairs is increased beyond 10-20 to achieve genome-wide SNP genotyping, then broader genomic coverage is achieved, but amplification yield is compromised by accumulation of non-target amplicons
Solution Approach 1:
The blocking oligonucleotide acts as a selective mediator that enables the use of large numbers of primer pairs (100-1000+) by specifically targeting and suppressing non-target amplicons. This allows genome-wide SNP genotyping to be performed with high genomic coverage while maintaining sufficient amplification yield of true target sequences, as the blocking oligonucleotide prevents the harmful accumulation of non-specific products that would otherwise dominate the reaction.
Solution Approach 2:
The invention extracts or removes non-target amplicons from the reaction mixture by introducing blocking oligonucleotides that specifically bind to them, followed by selective degradation methods. This extraction approach separates the harmful non-target products from the valuable target amplicons, allowing the latter to be analyzed with high confidence even when using a large number of primer pairs for comprehensive genomic coverage.
3Measurement precision
If careful attention is paid to primer design to reduce non-target amplicons, then specificity of amplification is improved, but the number of primer pairs that can be used in a single multiplex reaction is still limited to 10-20
Solution Approach 1:
The blocking oligonucleotide serves as a secondary intermediary that works in conjunction with carefully designed primers. While primer design remains important for initial specificity, the blocking oligonucleotide provides an additional layer of protection by specifically targeting non-target amplicons that escape primer specificity controls. This dual approach enables the use of many more primer pairs (100-1000+) while maintaining high amplification specificity, effectively removing the 10-20 primer pair limitation.
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 significantly reduces non-target amplicons to 50% or less by weight, enabling efficient and accurate amplification of multiple target sequences without compromising yield, even with a large number of primer pairs, thus enhancing the utility of amplification products for analysis.
Implementation Method 1
contacting a nucleic acid sample with two or more primer pairs for two or more target nucleic acid sequences under template dependent primer extension reaction conditions, e.g., polymerase chain reaction (PCR) conditions
Data Source
AI summary
Methods and compositions for performing low background multiplex nucleic acid amplification reactions are provided. Aspects of the invention include contacting a nucleic acid sample with two or more primer pairs for two or more target nucleic acids under template dependent primer extension reaction conditions, e.g., polymerase chain reaction (PCR) conditions. The resultant amplified composition is then contacted with target nucleic acid circularizing reagents, and product circularized target nucleic acids are then selected, e.g., for further amplification. Also provided are systems and kits that find use in practicing embodiments of the inventions.