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Method for growing DNA at 3'-OH terminal of RNA based on terminal deoxyribonucleotide transferase (TdT) and free of template dependence

A -OH, template-free technology, applied in the biological field, can solve the problems of low expression, unfavorable miRNA, poor stability, etc., and achieve the effect of easy degradation

Inactive Publication Date: 2020-04-07
TIANJIN UNIV
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Problems solved by technology

However, because miRNA has the characteristics of short sequence, low expression level and relatively poor stability, reverse transcription real-time quantitative PCR technology usually requires complex primer design or primer modification, which is unfavorable for sensitive and universal detection of miRNA

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  • Method for growing DNA at 3'-OH terminal of RNA based on terminal deoxyribonucleotide transferase (TdT) and free of template dependence
  • Method for growing DNA at 3'-OH terminal of RNA based on terminal deoxyribonucleotide transferase (TdT) and free of template dependence
  • Method for growing DNA at 3'-OH terminal of RNA based on terminal deoxyribonucleotide transferase (TdT) and free of template dependence

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Embodiment Construction

[0036] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited thereto.

[0037] Concrete steps of the present invention are as follows:

[0038] (1) Different raw materials, different times, and different RNA concentrations

[0039] Add the raw materials in the order of dATP, dTTP, dGTP, dCTP and dNTPs, and observe which base is preferentially extended by TdT when RNA is used as a primer, such as figure 1 As shown, dNTPs have the best elongation efficiency. Among them, it is compared with the extended DNA.

[0040] Secondly, using different reaction times, including 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 90min, 120min as a gradient, observe the situation of RNA extension at different times, like figure 2 As shown, the length and amount of extension are linear with time. Use different RNA concentrations, including 2μM, 1μ...

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Abstract

The invention discloses a method for growing DNA at the 3'-OH terminal of RNA based on TdT and free of template dependence. The method mainly comprises the following steps: with miRNA122 and DNA122 asprimers, adding dNTPs, 1*CoCl2, 1*RB10* and TdT to form a 20-microliter reaction system; adjusting reaction conditions including reaction time, reaction concentration, reaction raw materials and reaction substrates; verifying a RNA extension effect through polyacrylamide gel electrophoresis and a silver staining method; and verifying the difference of extension effects of DNA and RNA under the action of the TdT through a fluorescence method. The invention relates to the novel application of TdT; and a series of high-sensitivity RNA detection platforms can be established by utilizing the new application, and a new detection direction is provided for miRNAs such as tumor markers in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically based on the fact that TdT enzyme can add dNTPs to the 3'-OH end of RNA, thereby forming a structure of growing DNA after RNA, and providing a new strategy for RNA detection. 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. [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 confirmed that the early diagnosis of cancer can effectively improve the survival rate of cancer, and the sensitive detection of miRNA ...

Claims

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Application Information

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IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 常津崔警予宫晓群韩厚玉朴佳芳
Owner TIANJIN UNIV
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