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Self-transcription sequence detection method and application, and eukaryon controlling element identification method and application

A technology of detection method and identification method, which is applied in the field of detection of self-transcribed sequences, can solve problems such as inability to add polyA, low reliability, amplification, etc., and achieve the effects of avoiding a large number of losses, overcoming missed detection, and improving reliability

Inactive Publication Date: 2019-10-22
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the actual situation is that a considerable proportion of the insert fragments themselves will have a pA site, and in this case, when the tail is added after transcription, polyA cannot be added to the pA site after the original expected general sequence, but The tailing signal that comes with the insert will be used to add before the universal sequence downstream of the insert, which makes it impossible for the reverse transcription primers used in the existing method to reverse transcribe and amplify the mRNA of this type of insert
Therefore, this kind of detection method may lose a lot of information in the actual detection process, which makes the reliability of the obtained results low.

Method used

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  • Self-transcription sequence detection method and application, and eukaryon controlling element identification method and application

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Embodiment 1

[0034] The following combination figure 1 Explain the principle of the present invention, figure 1 It is a schematic diagram of the principle of the present invention. Such as figure 1 Shown:

[0035] (1) Genomic DNA is randomly fragmented to select an appropriate size and repaired, and processed to obtain a gene library of inserted fragments. According to the promoter-ORF sequence-intron (not shown in the figure)-inserted DNA fragment, the reporter vector was constructed, wherein the turning arrow was the promoter, and there were general sequences and pA sites downstream of the inserted DNA fragment.

[0036] (2) Initiate transcription, if the candidate sequence of the eukaryotic regulatory element inserted by the inserted DNA fragment has a silencer function, it will inhibit the transcriptional activity of the promoter, resulting in a decrease in the copy number of the transcribed RNA, followed by reverse transcription There was no positive correlation between copy numbe...

Embodiment 2

[0039] Respectively according to the traditional reverse transcription downstream primer using the complementary sequence of the universal sequence and using the method in Example 1 to construct the reverse transcription sequence for 6 random bases and 18 thymine T and the universal amplification primer sequence from the 3' end Transcription downstream primers are used to construct the Drosophila genome reverse transcription library according to the conventional library construction method. For the reverse transcription library of some genes, see figure 2 . figure 2 It is a partial comparison result of the Drosophila reverse transcription signal in the comparison experiment of another embodiment of the present invention. Such as figure 2 As shown, the top is the reverse transcription signal measured by the traditional method, the middle is the reverse transcription signal measured by the method provided in Example 1, and the bottom is the signal intensity difference obtain...

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Abstract

The invention discloses a self-transcription DNA sequence detection method and application capable of reducing information loss and improving reliability, and a eukaryon controlling element identification method and application. The detection method comprises the following steps that a reporter vector is constructed and comprises a promoter, an ORF sequence and an inserted DNA segment which are arranged in sequence; the reporter vector is transcribed into mRNA; a reverse transcription primer is used for reversely transcribing the mRNA to obtain cDNA; the reverse transcription primer comprises5'-Z-Tm-Xn-3'. The detection method adopts the new reverse transcription primer for reversely transcribing the mRNA obtained in the transcription process. Compared with the prior art, the method can also realize good transcription of mRNA with a poly A sequence added to a self-contained pA locus of an insertion segment, complete information transfer of the insertion segment in the detection process can be ensured, loss of a large amount of information is avoided, therefore the reliability of the detection result is improved, and the detection omission phenomenon is effectively avoided.

Description

technical field [0001] The invention relates to the technical field of genomics, in particular to a detection method and application of an autotranscribed sequence, an identification method and the application thereof. Background technique [0002] The completion of the Human Genome Project opened up a comprehensive and in-depth study of the function of DNA sequences. However, for up to 98% of the non-coding DNA in the human genome sequence, the relevant functional research progress is slow, and the functional understanding of this part of the genome sequence is seriously lacking. How to analyze and determine the function of genome sequences is a major problem in the field of genomics, and the solution of this problem will also lay a solid foundation for synthetic biology and other practical applications. [0003] The important types of regulatory elements in the eukaryotic genome mainly include enhancers, silencers, insulators, and promoters. At present, the research on en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2521/107C12Q2531/113C12Q2535/122
Inventor 侯春晖何娜牛龙见姜大新
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA