MicroRNA Detection via PolyA Tailing and Cpf1
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Solution Overview
Problem
Current methods for detecting microRNAs are time-consuming, costly, and lack specificity and sensitivity due to the short sequence length and high sequence similarity among miRNA family members, making it difficult to directly detect miRNAs using CRISPR-Cas systems.
Innovation Solution
A microRNA detection method and kit that combines poly-adenine (PolyA) tailing with the CRISPR-Cpf1 system, where a PolyA sequence is added to miRNAs to enhance amplification and recognition by Cpf1, allowing for more stable and specific detection through the use of a fluorescence-labeled single-stranded DNA probe.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional detection methods (Northern Blot, RT-qPCR, sequencing) are used, then detection accuracy is achieved, but detection time is long and costs are high
Solution Approach 1:
The detection process is divided into distinct modular steps: PolyA tailing, cDNA synthesis, DNA amplification, and Cpf1 detection. Each step can be independently optimized and performed, allowing parallel processing and reducing overall detection time while maintaining accuracy through systematic quality control at each stage.
Solution Approach 2:
The invention introduces intermediate conversion steps (PolyA tailing and cDNA synthesis) that transform the original miRNA into a form more suitable for amplification and detection. This intermediary process enables the use of highly specific Cpf1 detection while reducing direct detection time, as the intermediate DNA form allows for faster and more specific recognition.
2Measurement precision
If CRISPR-Cas systems are used for miRNA detection, then specificity is improved, but detection efficiency is low due to short miRNA sequence length and few PAM sites
Solution Approach 1:
The invention performs preliminary PolyA tailing on miRNAs before CRISPR detection, adding multiple adenine residues to the 3' end of miRNAs. This preliminary action creates additional PAM sites (TTTN sequences) that Cpf1 can recognize, thereby improving detection efficiency without compromising the specificity of CRISPR-Cas system.
Solution Approach 2:
The invention changes the physical-chemical parameters of the miRNA by adding PolyA tails, which alters the sequence composition and length. This parameter change increases the number of available PAM sites for Cpf1 binding, enabling more efficient detection while maintaining the original miRNA's specific recognition capability through the crRNA guide.
3Measurement precision
If miRNA amplification is performed, then detection sensitivity is improved, but detection cost increases
Solution Approach 1:
The invention uses a disposable PolyA tailing reaction that adds universal adenine tails to all miRNAs in the sample. This single-step modification enables subsequent amplification and detection of multiple miRNAs simultaneously, reducing per-sample costs compared to traditional methods that require separate reactions for each miRNA target.
Solution Approach 2:
The PolyA tailing step serves multiple functions: it amplifies the signal for detection, creates PAM sites for Cpf1 recognition, and works universally for all miRNA sequences regardless of their original composition. This multi-functionality reduces the need for multiple specialized reagents and procedures, thereby lowering overall detection costs while improving sensitivity.
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 improves detection efficiency, reduces costs, and enhances sensitivity and specificity, enabling rapid, accurate, and cost-effective detection of miRNAs, suitable for clinical and diagnostic applications.
Implementation Method 1
adding a poly-adenine (PolyA) sequence of at least two adenines to a microRNA of a sample
Implementation Method 2
synthesizing a cDNA based on a tailed microRNA
Implementation Method 3
recognizing the cDNA by Cpf1
Implementation Method 4
activating Cpf1 to cleave a probe for detection
Implementation Method 5
a fluorescence-labeled single-stranded DNA probe
Data Source
AI summary
The present invention discloses a microRNA detection method and kit for rapid detection of microRNA nucleic acids in biological and environmental samples, including microRNA tailing and a Cpf1 detection system suitable for rapid detection. The present invention is the first to use a combination of microRNA tailing and Cpf1 detection to detect microRNAs, with the advantages of high sensitivity, strong specificity, short time consumption, high throughput, direct interpretation by the naked eye, no dependence on large-scale experimental equipment and the like. These advantages make the detection method developed by the present invention convenient for rapid detection, and identification and diagnosis of microRNAs in biological and environmental samples at a clinical front line.