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Reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase and used for detecting micro RNA

A deoxynucleotide and transferase technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve the problems of inability to catalyze the polymerization of deoxyribose triphosphate, short targets, etc., and achieve good detection specificity. Effect

Pending Publication Date: 2019-07-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first step is the short nucleic acid extension mediated by TdT enzyme: when there is no hydroxyl group at the 3' end of the nucleic acid, TdT enzyme cannot catalyze the polymerization reaction of deoxyribose triphosphate; when miRNA is present, TdT enzyme will extend at its 3' end Generate a DNA strand to solve the problem of the target being too short

Method used

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  • Reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase and used for detecting micro RNA
  • Reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase and used for detecting micro RNA
  • Reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase and used for detecting micro RNA

Examples

Experimental program
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Effect test

Embodiment 1

[0060] (1) TdT extension step: first prepare 2 , 10pM DNA-122 (sequence shown in SEQID No.4), 1×TdT reaction buffer and 10U TdT enzyme reaction system, then react at a constant temperature of 37°C for 30min, and then inactivate at 85°C for 25min.

[0061] (2) Reverse transcription step: Prepare a reaction system containing 10-fold diluted reaction product from the previous step, 100 nM primer I (sequence shown in SEQ ID No.1), 1× reverse transcription buffer, and reverse transcriptase MixI, Then react at a constant temperature of 37°C for 15min, followed by inactivation at 85°C for 5s.

[0062] (3) Real-time fluorescent quantitative PCR: the reaction is carried out on a 96PCR plate, and the reverse transcription product diluted 10 times, 250nM primer II (sequence shown in SEQ ID No.2), 250nM primer III (sequence As shown in SEQ ID No.3) and the reaction system of 1×SYBR Premix ExTMTaqⅡ, then put the plate into 480InstrumentII for PCR reaction. The procedure is to heat at 9...

Embodiment 2

[0065] (1) TdT extension step: first prepare 2 , 10pM DNA-122, 1×TdT reaction buffer and 10U TdT enzyme reaction system, then react at a constant temperature of 37°C for 30min, and then inactivate at 85°C for 25min.

[0066] (2) Reverse transcription step: Prepare a reaction system containing 10-fold diluted reaction product from the previous step, 100nM primer I, 1× reverse transcription buffer and reverse transcriptase MixI, then react at a constant temperature of 37°C for 15min, then 85°C Inactivate 5s.

[0067] (3) Real-time fluorescent quantitative PCR: The reaction was carried out on a 96PCR plate. Firstly, a reaction system containing 10-fold diluted reverse transcription product, 250nM primer II, 250nM primer III and 1×SYBR Premix ExTMTaqII was prepared in the well, and then the plate was put into a 480Instrument II for PCR reactions. The procedure is to heat at 95°C for 30s, then cycle through heating at 95°C for 5s and extension at 63°C for 30s, and terminate the...

Embodiment 3

[0070] (1) TdT extension step: first prepare 2 , 10pM DNA-122, 1×TdT reaction buffer and 10U TdT enzyme reaction system, then react at a constant temperature of 37°C for 30min, and then inactivate at 85°C for 25min.

[0071] (2) Reverse transcription step: Prepare a reaction system containing 10-fold diluted reaction product from the previous step, 100nM primer I, 1× reverse transcription buffer and reverse transcriptase MixI, then react at a constant temperature of 37°C for 15min, then 85°C Inactivate 5s.

[0072](3) Real-time fluorescence quantitative PCR: The reaction was carried out on a 96PCR plate. Firstly, a reaction system containing 10-fold diluted reverse transcription product, 250nM primer II, 250nM primer III and 1×SYBR Premix ExTMTaqII was prepared in the well, and then the plate was put into a 480Instrument II for PCR reactions. The procedure is to heat at 95°C for 30s, then cycle through heating at 95°C for 5s and extension at 63°C for 30s, and terminate the...

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Abstract

The invention discloses a reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting microRNA, in particular to a reverse transcription PCR detection method of aliver injury microRNA marker miR-122, and belongs to the technical field of biology. Specifically, the method utilizes the function that TdT catalyzes RNA to extend out of single-stranded DNA so as toachieve miR-122 elongation; and then reverse transcription is carried out on the extension product by using a universal primer and rapid amplification is carried out by real-time quantitative PCR, sothat sensitive and specific quantitative detection on trace miRNA in a pure liquid phase system is achieved. The method has good specificity, sensitivity and anti-interference capability, and provides a new direction for establishing a sensitive and specific miRNA detection method.

Description

technical field [0001] The invention discloses a reverse transcription PCR method mediated by terminal deoxynucleotidyl transferase for detecting microRNA, belonging to the field of biotechnology. Background technique [0002] In recent years, many microRNAs (microRNAs, miRNAs) have become potential tumor markers for early diagnosis of cancer. Among the known miRNAs, 50% are related to a variety of human tumors, and the abnormal expression of these miRNAs is closely related to the occurrence and development of tumors. Among them, liver cancer (hepatocellular carcinoma, HCC) had approximately 850,000 new cases and 810,000 fatal cases in the world in 2015, and its morbidity and mortality rates are among the top. [0003] Due to the rapid increase in the number of cancer patients in my country, scientific prevention, early diagnosis, timely treatment and postoperative monitoring of cancer have become important research topics and directions. Many biological studies have confi...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2521/107C12Q2521/131
Inventor 常津朴佳芳宫晓群王时超崔警予
Owner TIANJIN UNIV
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