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System detecting method for DNA (deoxyribonucleic acid) transcription direction and transcription template and application thereof

A technology of transcription direction and detection method, which is applied in the field of system detection of DNA transcription direction and transcription template, can solve the problems of cumbersome northernblot steps, high cost of single-stranded probes, and high cost, and achieves low price, low cost and high speed. Effect

Active Publication Date: 2014-09-03
SOUTH CHINA AGRI UNIV
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004]Currently, methods for detecting gene transcription direction mainly include northern blot, high-throughput sequencing, etc. However, the steps of northern blot are cumbersome, and the entire experimental process takes 2 days to complete , and if the purpose is to detect gene transcription direction and template, the cost of synthesis and labeling of single-stranded probes used in hybridization 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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  • System detecting method for DNA (deoxyribonucleic acid) transcription direction and transcription template and application thereof
  • System detecting method for DNA (deoxyribonucleic acid) transcription direction and transcription template and application thereof
  • System detecting method for DNA (deoxyribonucleic acid) transcription direction and transcription template and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[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), marker D2000 (Genestar), reverse transcription kit (Promega), RNase inhibitor (Thermo), S1 nuclease (TaKaRa), Trizol (TaKaRa), DNase I (Thermo).

[0071] 5...

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 Vector NTI and primer premier5 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 appropriate annealing temperature and reaction conditions for the primers. 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 transcr...

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Abstract

The invention provides a system detecting method for a DNA (deoxyribonucleic acid) transcription direction and a transcription template and application thereof. The method is mainly used for detecting the transcription direction of a target gene by utilizing the character that S1 nuclease can degrade single-stranded DNA; a specific RT-PCR (reverse transcription-polymerase chain reaction) product cDNA (complementary deoxyribonucleic acid) of a to-be-detected gene strand is taken as a template, PCR (polymerase chain reaction) is carried out by utilizing a designed gene upstream primer or downstream primer R, and judgment about whether a transcription template of the to-be-detected gene is a positive chain or a negative chain of DNA is carried out according to the PCR product electrophoresis result. The method disclosed by the invention can be used for analyzing whether a transcription process from DNA to mRNA (messenger ribonucleic acid) is single-way transcription or double-way transcription; for the single-way transcribed DNA segment, the method can further determine that the template chain of DNA transcription is the positive chain or the negative chain of DNA. Besides, the method can complete detection within about 12 hours, has characteristics of being simple to operate, quick in speed, and low in cost, is developed into a detection kit, and can be widely popularized and applied in scientific research and novel gene function research.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a systematic detection method of DNA transcription direction and transcription template and its application. Background technique [0002] In the field of molecular biology, gene transcription is a process in which a strand of DNA is used as a template to synthesize RNA under the action of RNA polymerase. Which strand of the DNA duplex is used as a template for transcription varies with different genes or DNA fragments. In recent years, studies have found that both strands of the DNA region of some genes can be transcribed. Transcription using a single strand of DNA as a template is unidirectional transcription, and the transcribed cDNA is a single strand; while there is a gene transcription method in which both strands of the DNA region can be transcribed, we call it bidirectional transcription, and the transcribed cDNA is a double chain. In the er...

Claims

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Application Information

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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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