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A systematic detection method of dna transcription direction and transcription template and its application

A detection method and technology of transcription direction, applied in the field of systematic detection of DNA transcription direction and transcription template, can solve the problems of cumbersome steps in northern blot, high cost and high cost of single-stranded probes, and achieve low price, low cost and fast speed Effect

Active Publication Date: 2016-03-30
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for detecting gene transcription direction mainly include northern blot, high-throughput sequencing, etc. However, the steps of northern blot are cumbersome, and the whole experimental process takes 2 days to complete. The cost of synthesis and labeling of single-stranded probes is very high
High-throughput sequencing can also detect the direction of gene transcription. However, high-throughput sequencing is an omics research method, and the research object is the entire transcriptome or genome. The cost is very high, and it takes at least one month to complete
Such time-consuming and costly high-throughput sequencing is very unsuitable for studying the transcription direction and template of a single gene or a few genes

Method used

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  • A systematic detection method of dna transcription direction and transcription template and its application
  • A systematic detection method of dna transcription direction and transcription template and its application
  • A systematic detection method of dna transcription direction and transcription template and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Analysis of mouse β-actin and Tsix gene transcription direction

[0064] 1. Test materials and test methods

[0065] (1) Test animals

[0066] Kunming white mice, female mice 7-8 weeks old, male mice 3-6 months old, feeding conditions: light 6:00AM-8:00PM, temperature 20-25℃, free to eat.

[0067] (2) Main equipment and instruments

[0068] Scissors, tweezers, and absorbent cotton should be treated with RNase inhibitors before use; pipette tips, centrifuge tubes, and PCR tubes should all be treated with DECP water, and the concentration of DECP should be 0.1%. Ice maker, -20℃ refrigerator, -80℃ refrigerator, desktop centrifuge, high-speed refrigerated centrifuge, electrophoresis instrument, electronic analytical balance, PCR instrument.

[0069] (3) Experimental reagents

[0070] Mix (Transgene), markerD2000 (Genestar), reverse transcription kit (Promega), RNase inhibitor (Thermo), S1 nuclease (TaKaRa), Trizol (TaKaRa), DNase I (Thermo).

[0071] 50×TAE...

Embodiment 2

[0139] The total RNA in the chicken liver tissue was extracted and reverse-transcribed, and the method was the same as in Example 1. Use the software VectorNTI and primerpremier5 to design the primers GHR (LRP1)-F3 (sequence shown in SEQ ID NO.5) and GHR (LRP1)-R3 (sequence shown in SEQ ID NO.6) of the target gene GHR, and determine the suitable annealing temperature of the primers and reaction conditions. The primer information is shown in Table 8:

[0140] Table 8 Chicken GHR primer information

[0141]

[0142] GHR gene PCR reaction conditions are as follows:

[0143] 95°C, 5min→(94°C, 30sec→60°C, 30sec→72°C, 30sec) 40cycles→72°C, 5min→16°C, 1h.

[0144] Subsequent detection methods and operations are the same as in Example 1.

[0145] Experimental results such as Figure 11~13 shown. According to our experimental results, it is determined that the detected DNA segment is bidirectionally transcribed, and both strands of DNA can be used as templates to transcribe RN...

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Abstract

The invention provides a method for systematically detecting DNA transcription direction and transcription template and its application. This method mainly uses the property of S1 nuclease to specifically degrade single-stranded DNA to detect the transcription direction of the target gene; using the cDNA of the specific RT-PCR product of the gene chain to be detected as a template, the upstream primer F or downstream primer R of the designed gene is used to carry out PCR, according to the electrophoresis results of PCR products, it is judged whether the transcription template of the gene to be tested is the positive strand or the negative strand of DNA. This method can be used to analyze whether DNA is transcribed in one direction or bidirectionally in the process of mRNA transcription; for a DNA fragment transcribed in one direction, it can further determine whether the template strand for DNA transcription is the positive strand or the negative strand of DNA. In addition, this method can complete the detection in about 12 hours, and has the remarkable characteristics of simple operation, fast speed, and low cost. It can be developed into a detection kit and can be widely popularized and applied in scientific research and new gene function research.

Description

Technical field [0001] The invention is a molecular biotechnology field.More specifically, it involves a system detection method and application of a DNA transcription direction and the transcription template. Background technique [0002] In the field of molecular biology, gene transcription is based on a chain of DNA as a template, and the process of synthesizing RNA under the action of RNA polymerase.Which chain in the DNA dual chain is different from different genes or DNA fragments.In recent years, studies have found that some genes of DNA areas can be transcribed.The transcription of a single chain with DNA is the one -way transcription, and the CDNA obtained by the transcript is a single chain; and the two chains that exist in the DNA area can be transcribed.For dual chain.In the era of high -throughput sequencing, a large number of genes or DNA fragments have been found to have antonym transition books, and many genes are two -way transcription. [0003] The transcription...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2521/307C12Q2565/125
Inventor 张丽张细权聂庆华
Owner SOUTH CHINA AGRI UNIV
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