CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage
| Autor: | Tian Tian, Jing Lv, Erhu Xiong, Zhenzhen Zhang, Yongzhong Jiang, Yijuan Bao, Xiaoming Zhou, Yang Li |
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| Rok vydání: | 2020 |
| Předmět: |
Materials science
Inverted repeat Loop-mediated isothermal amplification Bioengineering 02 engineering and technology Cleavage (embryo) 01 natural sciences Humans CRISPR Free loop Instrumentation Fluid Flow and Transfer Processes Cas9 Process Chemistry and Technology 010401 analytical chemistry Contamination 021001 nanoscience & nanotechnology Combinatorial chemistry eye diseases 0104 chemical sciences Protospacer adjacent motif Molecular Diagnostic Techniques sense organs CRISPR-Cas Systems 0210 nano-technology Nucleic Acid Amplification Techniques |
| Zdroj: | ACS Sensors. 5:1082-1091 |
| ISSN: | 2379-3694 |
| DOI: | 10.1021/acssensors.0c00034 |
| Popis: | Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format. |
| Databáze: | OpenAIRE |
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