A method for detecting microRNA using two-step amplification method
A rolling circle amplification and purpose technology, which is applied in the fields of molecular biology and nucleic acid chemistry, can solve problems that cannot meet human needs, and achieve good anti-interference ability, good selectivity, and lower detection limit.
Inactive Publication Date: 2019-01-29
武汉顺可达生物科技有限公司 +1
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Problems solved by technology
With the development of the times, these three detection methods can no longer meet the needs of human beings, and more sensitive detection methods are urgently needed for the analysis and detection of actual samples.
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Embodiment 1
[0032] 1. Design of four primers for rolling circle amplification template, restriction endonuclease cleavage site complementary sequence and circle-mediated isothermal amplification in the present invention
[0033] (1) if figure 1 As shown, the DNA was synthesized by Sangon Company, and the rolling circle amplification template was designed for the sequence of microRNA 21 (5'-uagcuuaucagacugauguuga-3'), which is an important tumor marker. The template for rolling circle amplification includes three parts, the 5' end and the 3' end respectively and half of the microRNA complementary region, which is responsible for recognizing the corresponding microRNA, forming a double-stranded structure of DNA-RNA hybridization and rolling circle amplification template The end-to-end is connected to form a circle, and the 5' end of the template is phosphorylated. The sequence of the rolling circle amplification template is 5'-PO 4 -CTG ATA AGC TAC GAC TCT AGA GGA TCCCCG GGT ACG TTT CCG T...
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The invention relates to a method for utilizing a rolling ring amplification-ring mediating isothermal amplification two-step amplification method for detecting MicroRNA(ribonucleic acid). According to the method, at first, a linear rolling ring amplification template with target microRNA as a cyclized connecting probe is designed, rolling ring amplification is carried out with the linear rolling ring amplification template as a primer, and first-step amplification of signals is achieved; a rolling ring amplified product is cut into the same short single chains through restriction incision enzymes, ring mediating amplification is carried out with the short single chains as templates, and second-step amplification of the signals is achieved. The amplification process of ring mediating isothermal amplification is monitored through a real-time fluorescence quantification PCR(polymerase chain reaction), a DNA color developing agent is added into an amplified sample, when amplified DNA products are increased, fluorescence of the DNA color developing agent is enhanced, and the fluorescence signals are collected and output by the real-time fluorescence quantification PCR. By adopting the two-step amplification method for amplifying the signals of microRNA, the method for detecting microRNA is low in background and high in sensitivity.
Description
technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for detecting MicroRNA using a rolling circle amplification-circle-mediated isothermal amplification two-step amplification method. Background technique [0002] MicroRNAs (microRNAs) are endogenous non-coding small RNAs of about 21-23 bases, about 21-22 nucleotides in length, and their length can also vary from 19 to 25 nucleotides . MicroRNA is generally produced in the body by a two-step enzymatic hydrolysis process. The most primitive is pri-microRNA, which is about 300-1000 bases in length; after pri-microRNA is processed once, it becomes pre-microRNA, the precursor of microRNA, with a length of about The length is 70-90 bases; the pre-microRNA is digested by Dicer enzyme to become a mature microRNA with a length of about 20-24 nt. The specific principle is as follows: pri-microRNA is digested by Drosha enzyme to obtain a pre-microRNA wit...
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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2525/131C12Q2531/125C12Q2531/119C12Q2561/113C12Q2563/107
Inventor 周翔陈玉琪宋燕燕王少儒
Owner 武汉顺可达生物科技有限公司