CN107604043B - The high throughput identification method of genome insulator - Google Patents
The high throughput identification method of genome insulator Download PDFInfo
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- CN107604043B CN107604043B CN201710659482.5A CN201710659482A CN107604043B CN 107604043 B CN107604043 B CN 107604043B CN 201710659482 A CN201710659482 A CN 201710659482A CN 107604043 B CN107604043 B CN 107604043B
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Abstract
The invention discloses the high throughput identification methods of genome insulator, comprising: 1) construct report carrier: report carrier successively has promoter, the area ORF, introne and insulator candidate sequence insert district and enhancer；Enhancer is used to enhance the expression of upstream sequence, and insulator candidate sequence insert district is inserted with insulator candidate sequence；2) report carrier is expressed and is transcribed；3) situation is transcribed after detecting the editor of transcription sequence, determines whether insulator candidate sequence is insulator.The report carrier library of this method building can cover 90% to 99% Genome noncoding regions, do not need protein positions information, all analyze；Constructed library is transferred into the cell, can detect whether almost whole gene group sequence has insulator active simultaneously, can be analyzed with large-scale parallel, avoid single, a small amount of analysis；Because constructed library covers nearly all site, zone of ignorance can equally be detected analysis.
Description
Technical field
The present invention relates to genomics, in particular to the high throughput identification method of genome insulator.
Background technique
It with the completion of the Human Genome Project, the formation of accurate medical concept and graduallys mature, people are for genome
The knowledge of function itself has a more and more comprehensive requirement, however actually scientist for genome functions understanding but much
The demand of reality is lagged behind, is difficult to really implement in concept so that current accurate medical treatment is more flowed, or implement
It is basic unstable.
In addition to gene, how there is a serious shortage of divide the functional knowledge about the non-coding sequence for accounting for human genome 98%
The potential function for analysing and determining genome sequence is the great theoretical question of biology, and the basis of application.Insulator regulation
Element is a kind of important DNA sequence dna of eukaryotic gene group, and enhancer member is isolated first is that passing through in their most important functions
Part, thus make genome realize specifically, selectively activation target gene.
The Functional Characterization of existing insulator is small-scale, the analysis of scattered a small amount of DNA sequence dna；Large-scale insulator
The identification technology of function element has not been reported, the method only predicted by binding protein, and such methods are not because be root
According to the analysis that function carries out, therefore there is greatly uncertain and unreliability.Its deficiency is mainly manifested in following 3 sides
Face:
First, rely on existing insulator protein binding site information to predict, it, then can not be pre- such as without protein binding information
It surveys；
Second, whether the site predicted is functional, can only individually measure, can not large scale analysis；
Whether third has isolation subfunction to the non-isolated protein bound region of son, can neither predict, can not divide
Analysis.
Insulator how is efficiently identified to be a problem to be solved.
Summary of the invention
The purpose of the present invention is to provide a kind of high throughput identification methods of genome insulator.
The technical solution used in the present invention is:
A method of identification genome insulator, comprising:
1) construct report carrier: successively there is report carrier promoter, the area ORF, introne and insulator candidate sequence to insert
Enter area and enhancer；Enhancer is used to enhance the expression of upstream sequence, and insulator candidate sequence insert district is waited inserted with insulator
Select sequence；
2) report carrier is expressed and is transcribed；
3) situation is transcribed after detecting the editor of transcription sequence, determines whether insulator candidate sequence is insulator.
As the further improvement of the above method, also inserted with RNA between insulator candidate sequence insert district and enhancer
Critical sequences.Further, RNA critical sequences are poly-A sequence.
As the further improvement of the above method, insulator candidate sequence passes through construction of gene library.
As the further improvement of the above method, report carrier is transferred to cell class in a manner of library and carries out expression transcription.
As the further improvement of the above method, promoter is selected from artificial synthesized promoter SCP.
As the further improvement of the above method, the area ORF is selected from fluorescence protein gene, colour developing enzyme gene.
As the further improvement of the above method, introne is selected from artificial synthesized intron sequences.
As the further improvement of the above method, enhancer is selected from virus SV40 enhancer, cmv enhancer.
The beneficial effects of the present invention are:
The method of the present invention has the advantages that have as follows:
First, the report carrier library of building can cover 90% to 99% Genome noncoding regions, not need albumen position
Confidence breath, is all analyzed；
Second, constructed library is transferred into the cell, can detect simultaneously almost whole gene group sequence whether have every
Ion activity can be analyzed with large-scale parallel, avoid single, a small amount of analysis；
Third, because constructed library covers nearly all site, zone of ignorance can equally be detected analysis.
Detailed description of the invention
Fig. 1 is the schematic illustration of the technology of the present invention；
Fig. 2 is the chromosome dyad schematic diagram of drosophila.
Specific embodiment
A method of identification genome insulator, comprising:
1) construct report carrier: successively there is report carrier promoter, the area ORF, introne and insulator candidate sequence to insert
Enter area and enhancer；Enhancer is used to enhance the expression of upstream sequence, and insulator candidate sequence insert district is waited inserted with insulator
Select sequence；
2) report carrier is expressed and is transcribed；
3) situation is transcribed after detecting the editor of transcription sequence, determines whether insulator candidate sequence is insulator.
As the further improvement of the above method, also inserted with RNA between insulator candidate sequence insert district and enhancer
Critical sequences.Further, RNA critical sequences are poly-A sequence.The protection sequence for stablizing RNA by increasing, can make
The longer time can be stabilized by transcribing obtained RNA, be operated convenient for subsequent identification.
As the further improvement of the above method, insulator candidate sequence passes through construction of gene library.
As the further improvement of the above method, report carrier is transferred to cell class in a manner of library and carries out expression transcription.
Library can be used existing method and be prepared, such as extracting and purifying genomic DNA, ultrasound interrupt and recycles 500~
The segment of 600bp magnitude range is cloned by way of homologous recombination after reparation into plasmid vector.A small amount of conversion large intestine bars
Bacterium determines that transformation efficiency reaches about 10^7~10^8/ μ g (clone's number/plasmid amount) high-volume conversion Escherichia coli afterwards, in liquid
It is risen in culture medium and collects bacterium logarithmic amplification early period and extract plasmid.Carried out for two generations with a small amount of plasmid amplification Insert Fragment area
The complexity (a large amount of repeat amplification protcols of plasmid-free) for determining plasmid library is sequenced and to genome level of coverage (more than 85% or more
It is high).The plasmid library for reaching requirement can be used to test in next step.
The sequence of promoter without particular/special requirement, as long as can normal promotor gene transcription.In view of promoter is come
The simplicity in source, as the further improvement of the above method, promoter is selected from artificial synthesized promoter SCP.
As the further improvement of the above method, the area ORF is selected from fluorescence protein gene, colour developing enzyme gene.It in this way can be square
Just according to chromogenic reaction, the chromogenic reaction of such as fluorescence or substrate tentatively judges whether insulator candidate sequence has isolation
Subfunction.
Intron sequences, can be naturally occurring without particular/special requirement, be also possible to it is artificial synthesized, as long as can transfected
Cell in be sheared.As the further improvement of the above method, introne is selected from artificial synthesized intron sequences.
The sequence of enhancer is without particular/special requirement, as long as can enhance the activity of promoter.In view of source and operation
Simplicity etc., as the further improvement of the above method, enhancer is selected from virus SV40 enhancer, cmv enhancer.
Insulator candidate (insulator candidate sequence) is the genomic fragment of radom insertion, this needs 10^8-
In the identical carrier framework of independent segments insertion rest part of 9 (100,000,000 to billions of), to construct covering whole gene group
The report carrier library in non-duplicate area.
Below with reference to Fig. 1, the principle of the technology of the present invention is further illustrated.
When candidate sequence does not have insulator activity, by ORF sequence-Intron (introne)-insulator candidate sequence-
Poly A site sequence can transcribe under the action of enhancer, and the RNA copy and vector copies for transcribing out are positively correlated,
Introne can be cut off after transcription, be accordingly changed into the state of dotted arrow direction, transcribed edited sequence；
If candidate sequence has insulator activity, it is (red to the activation of starting region that downstream enhancer can be obstructed
Shown in STOP label), thus the RNA copy number decline for coming out transcription.
Because insulator candidate sequence can exist inside the RNA for transcribing out, it is equivalent to and self transcribes and serve as
The sequence mark of oneself, so as to know the one-to-one relationship between RNA and DNA vector.
Turned report carrier DNA is isolated and purified, the RNA after isolating and purifying transcription shearing, building can be used for extensive
The library of sequencing, sequencing, and analyze, realize high-throughput insulator identification.
Below with reference to experiment, technical solution of the present invention is further illustrated.
As shown in Fig. 2, top yl moiety is No. 2 chromosome R arm 5172760-5193360 of drosophila gene group for being analyzed
Gene within the scope of this 20kb, centre are the copy number of RNA sequencing, and height shows what corresponding region transcribed out from carrier
How much, the copy number in the region is corresponded to for insulator candidate sequence in carrier below, comparing RNA and candidate sequence copy number can be with
It was found that there is extraordinary positively related relationship in most of region, but the position among dotted line is erected in two red, carrier is waited
The copy number of sequence is selected to be apparently higher than the RNA copy number for transcribing out, this explanation hinders in report carrier containing this section of sequence
Break the activity of downstream enhancer, so as to cause transcription decline, so that the RNA copy number of corresponding this section of sequence is decreased obviously.
The experimental results showed that method of the invention can identify the insulator in genome well.
Claims (8)
1. a kind of method for identifying genome insulator, comprising:
1) construct report carrier: report carrier successively have promoter, the area ORF, introne, insulator candidate sequence insert district,
RNA critical sequences and enhancer；Enhancer is used to enhance the expression of upstream sequence, insulator candidate sequence insert district inserted with every
Ion candidate sequence；
2) report carrier is expressed and is transcribed；
3) situation is transcribed after detecting the editor of transcription sequence, determines whether insulator candidate sequence is insulator.
2. according to the method described in claim 1, it is characterized by: RNA critical sequences are poly-A sequence.
3. according to the method described in claim 1, it is characterized by: insulator candidate sequence passes through construction of gene library.
4. according to the method described in claim 1, it is characterized by: report carrier is transferred to cell class carry out table in a manner of library
Up to transcription.
5. according to the method described in claim 1, it is characterized by: promoter is selected from artificial synthesized promoter SCP.
6. according to the method described in claim 1, it is characterized by: the area ORF is selected from fluorescence protein gene, colour developing enzyme gene.
7. according to the method described in claim 1, it is characterized by: introne is selected from artificial synthesized intron sequences.
8. according to the method described in claim 5, it is characterized by: enhancer is selected from virus SV40 enhancer, cmv enhancer.
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| CN101001951A (en) * | 2004-08-02 | 2007-07-18 | 巴斯福植物科学有限公司 | Method for isolation of transcription termination sequences |
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| CN101001951A (en) * | 2004-08-02 | 2007-07-18 | 巴斯福植物科学有限公司 | Method for isolation of transcription termination sequences |
Non-Patent Citations (1)
| Title |
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| Identification and Characterization of Enhancer-Blocking Insulators to Reduce Retroviral Vector Genotoxicity;Amy C. Groth等;《PLOS ONE》;20131031;第8卷(第10期);第2页右栏第3段至第3页左栏第1段、第8页右栏第1段和图6 |
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