Chemically Modified Primers for Selective mRNA Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current RNA amplification methods by reverse-transcription polymerase chain reaction (RT-PCR) face challenges with genomic DNA contamination, where even small amounts of DNA are exponentially amplified, leading to inaccurate results due to the inability to distinguish between mRNA and genomic DNA, especially when introns are too small to prevent genomic DNA amplification.
Innovation Solution
The method involves using chemically modified oligonucleotides that span exon-exon junctions, with nucleotides modified at the exocyclic amino group, to selectively amplify mRNA targets while minimizing the amplification of corresponding genomic DNA, employing a combination of RNA-directed and DNA-directed synthesis enzymes and a hot start protocol for enhanced specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNase is used to remove genomic DNA contamination, then DNA contamination is reduced, but the RNA template is degraded due to high temperature inactivation requirements
Solution Approach 1:
The patent extracts and removes the harmful DNase enzyme from the reaction system by using a hot start protocol where the polymerase is activated only after the DNAse has been inactivated by heat treatment, thereby eliminating the need for chemical removal methods that would compromise RNA integrity
Solution Approach 2:
The patent performs preliminary heat treatment to inactivate DNase before initiating the amplification reaction, ensuring that the DNAse is completely inactivated prior to RNA template exposure, thus preventing RNA degradation while maintaining DNAse removal
2Measurement precision
If primers are designed to flank introns to prevent genomic DNA amplification, then mRNA specificity is improved, but the method fails when introns are too small to prevent DNA amplification
Solution Approach 1:
The patent changes the parameter of primer design by using chemically modified primers with nucleotides modified at the exocyclic amino group, which alters the primer's binding characteristics to achieve preferential amplification of mRNA over genomic DNA regardless of intron size
Solution Approach 2:
The patent employs composite primer structures combining chemically modified nucleotides with standard primer sequences, creating a hybrid molecule that leverages both the specificity of sequence complementarity and the enhanced binding characteristics of modified nucleotides to achieve reliable mRNA amplification
3Productivity
If real-time PCR is used without electrophoresis to detect nucleic acids simultaneously, then productivity is improved, but the ability to distinguish between mRNA and genomic DNA is lost
Solution Approach 1:
The patent replaces the mechanical separation method of electrophoresis with a chemical modification approach using chemically modified primers that inherently distinguish between mRNA and genomic DNA through preferential amplification, eliminating the need for post-amplification separation steps
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 allows for the preferential amplification of mRNA over genomic DNA, achieving clinically acceptable analytical specificity, even in the presence of significant genomic DNA contamination, thereby providing accurate gene expression studies and quantitative results.
Implementation Method 1
hybridizing a first oligonucleotide to said mRNA target
Implementation Method 2
performing RNA-directed DNA synthesis using at least one enzyme capable of RNA-directed synthesis
Implementation Method 3
amplifying the product of step a) using said first oligonucleotide and a second oligonucleotide with at least one enzyme capable of DNA-directed DNA synthesis
Data Source
AI summary
The present invention relates to a method, oligonucleotides, reaction mixtures and kits for the selective amplification of a messenger RNA target comprising an exon-exon junction, using an oligonucleotide that comprises at least one nucleotide modified at the exocyclic amino group.