CN108396043A - A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA - Google Patents

A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA Download PDF

Info

Publication number
CN108396043A
CN108396043A CN201710067173.9A CN201710067173A CN108396043A CN 108396043 A CN108396043 A CN 108396043A CN 201710067173 A CN201710067173 A CN 201710067173A CN 108396043 A CN108396043 A CN 108396043A
Authority
CN
China
Prior art keywords
sequence
restriction enzyme
loop
preparation
stem structure
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710067173.9A
Other languages
Chinese (zh)
Inventor
樊春海
王丽华
李江
诸兵
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Applied Physics of CAS
Original Assignee
Shanghai Institute of Applied Physics of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institute of Applied Physics of CAS filed Critical Shanghai Institute of Applied Physics of CAS
Priority to CN201710067173.9A priority Critical patent/CN108396043A/en
Publication of CN108396043A publication Critical patent/CN108396043A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/34Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00Nucleic acids vectors
    • C12N2800/80Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Abstract

The invention discloses a kind of preparation method and applications of the ends 5' phosphorylation single stranded DNA.The preparation method includes the following steps:(1) it is inserted into the restriction enzyme site sequence of restriction enzyme at target DNA both ends, forms loop-stem structure precursor;(2) the loop-stem structure precursor that step (1) obtains is dissolved in buffer solution and is made annealing treatment, obtain loop-stem structure solution;(3) restriction enzyme enzymatic treatment is added in the loop-stem structure solution for obtaining step (2), so that restriction enzyme is inactivated after processing, obtains digestion products;(4) by digestion products recycling obtained by step (3) to get.Preparation method step of the present invention is simple, of low cost, and phosphorylation is efficient, and the ends gained 5' phosphorylation single stranded DNA is easy to characterize and purify, and efficiency can be significantly improved by being applied to gene editing, targeting positioning and/or regulation and control, be had broad application prospects.

Description

One kind 5 ' holds the preparation method and applications of phosphorylation single stranded DNA
Technical field
The invention belongs to biotechnologies, more particularly to a kind of system of the ends 5' phosphorylation single stranded DNA (5'P-ssDNA) Preparation Method and its application.
Background technology
In recent years, with CRISPR (Clustered regularly interspaced short palindromic Repeats) technology be representative gene editing technology of new generation due to its accurately targeting and ease for use, in Basic of Biology Immeasurable potentiality have been shown in research and biomedical applications.This kind of gene editing system typically utilizes in bacterium Naturally occurring defense mechanism realizes that the targeting to invader dna is sheared.In addition to the Cas9 series of restriction inscribes of CRISPR systems Except enzyme (also abbreviation restriction endonuclease), some gene editing systems for being based on other restriction endonucleases (such as Argonaute) also receive Extensive concern.Such restriction endonuclease helps core using the single stranded DNA of 5' phosphorylations as " guiding " (guide DNA, gDNA) Sour enzyme identifies target gene sequence and is sheared to it.Compared with CRISPR systems, this kind of system using short single stranded DNA rather than RNA is as guiding molecule, the plasmid without structure for rna expression, more easily artificial synthesized and direct transfection.Currently, quotient The single stranded DNA chemical synthesising technology of industry is highly developed and cheap so that using single stranded DNA (ssDNA) as drawing The gene editing system led in cost Billy with the CRISPR systems of RNA advantageously.Importantly, conventional CRISPR System requirements target area downstream has specific PAM sequences, and Argonaute systems may be used with nearly needle without similar limitation To any target sequence.
But it is different from the natural ends DNA, the ends single stranded DNA 5' of implied terms synthesis do not introduce phosphate group. In order to obtain the guiding DNA that can be used for gene editing system, it would be desirable to the 5' after being synthesized to chemically synthesized single stranded DNA Hold phosphorylation modification.Therefore, the efficiency of phosphorylation just becomes one of the key factor for influencing gene editing efficiency.Have at present It is certain studies have shown that the 5'-P that is obtained of chemical phosphorylation method of modifying provided by commercialized primer Composite service SsDNA is bad to the shear efficiency of target gene in gene editing application, and is repaiied with the kinase catalytic phosphorylation of T4 nucleic acid The problem of decorations are difficult to optimize there is also efficiency, modified outcome is difficult to characterize and purify.
In addition, the cleavage reaction of common classics DNA restriction enzyme enzymatics is the generally recognized in genetic engineering and acts on In DNA double chain, shearing product is also double-strand, therefore is not suitable for the 5' phosphorylations for being directly used in single stranded DNA.
Invention content
The technical problem to be solved by the present invention is to for overcome phosphorylation efficiency existing in the prior art is low, shear efficiency not A kind of good equal difficult preparation method and applications of the ends 5' phosphorylation single stranded DNA of offer.The preparation method has easy to operate, phosphorus It is acidified efficient, applied widely and the advantages that have a extensive future.
One of technical proposal that the invention solves the above-mentioned problems is:A kind of preparation method of the ends 5' phosphorylation single stranded DNA, Include the following steps:
(1) it is inserted into the restriction enzyme site sequence of restriction enzyme at target DNA both ends, forms loop-stem structure precursor, wherein The target DNA is located at the cyclic single strand region of loop-stem structure precursor, and the restriction enzyme site sequence is located at loop-stem structure precursor Stem's double-stranded region;
(2) the loop-stem structure precursor that step (1) obtains is dissolved in buffer solution and is made annealing treatment, obtain stem ring knot Structure solution;
(3) restriction enzyme enzymatic treatment is added in the loop-stem structure solution for obtaining step (2), makes restriction enzyme after processing Enzyme inactivates, and obtains digestion products;
(4) by digestion products recycling obtained by step (3) to get.
Preferably, restriction enzyme site sequence described in step (1) contains the identification sequence and restriction enzyme site of restriction enzyme, The restriction enzyme site of the restriction enzyme is detached with identification sequence;More preferably, restriction enzyme site is located at identification sequence downstream, because To the 5 ' terminal sequence of product in restriction enzyme site downstream, there is no limit loop-stem structure is cut off under restriction enzyme enzyme effect, is released for this The target DNA that the ends 5' carry phosphate group is put, i.e., the described restriction enzyme site sequence and the base of adjacent target DNA have following sequence Row:
5'-S1N1N2N3-3'
3'-S2N4N5N6-5';
The S1For the positive sequence of the identification sequence of restriction enzyme, the S2For the identification sequence of restriction enzyme The reverse sequence of row, the N1、N2For the reverse complementary sequence of 3 ' two bases in end of purpose DNA, N3-N6For purpose DNA's Base, the restriction enzyme site are located at N2And N3Between and S2And N4Between.
Preferably, the restriction enzyme site sequence also contains protection base sequence, wherein S15 ' end have protection base Sequence 1, S23 ' ends there is protection base sequence 2, protection base sequence 1 and 2 reverse complemental of protection base sequence;Preferably, The protection base sequence is the high sequence of the G/C content containing 3-6 base, and the wherein high protection base sequence of G/C content is used In ensuring that loop-stem structure formed, ensure shear efficiency;Further preferably, the protection base sequence 1 is:GCGC、GGGC Or CCCG.
The restriction enzyme is the restriction enzyme that the restriction enzyme site of this field routine is detached with identification sequence.Compared with Goodly, the restriction enzyme is BseGI/BstF5I/BtsCI, Bts α I/BtsI, BtsIMutI or Bse3DI/ BseMI/BsrDI enzymes, the identification sequence (underscore) and restriction enzyme site (arrow locations) of above-mentioned 4 groups of enzymes are respectively:
The present invention is by selecting restriction enzyme site to be located at the restriction enzyme of identification sequence downstream so as under restriction enzyme site There is no limit can be used for preparing the 5'P-ssDNA of almost arbitrary sequence to the product 5' terminal sequences of trip.
Buffer solution described in step (2) is that this field handles buffer solution conventional used in nucleotide sequence, usually selects limit What property restriction endonuclease supplier products specification processed was provided.
In a preferred embodiment of the present invention, restriction enzyme selects BseGI, and target DNA both ends are in restrictive Enzyme cutting BseGI identifies that the loop-stem structure precursor sequence of sequence is:5'-GCGCGGATG+ target DNAs 3 ' end two base it is anti- To complementary series N1N2+ target DNA+CATCCGCGC-3', the wherein GCGC of italic are protection base sequence.Wherein, the buffering Liquid is provided by BseGI enzyme supplier products specifications, contains following ingredients:MgCl210 μ of 5mM, Tris-HCl 10mM, DNA M;pH 8.0.The annealing is that this field is conventional;It is handled 5 minutes preferably, the annealing is 95 DEG C of thermal cycler, Then it is cooled to 4 DEG C rapidly, and is kept for 10 minutes.Restriction enzyme enzymatic treatment and restriction enzyme described in step (3) Inactivation is that this field is conventional;Preferably, the enzyme-activity unit is 8-15U, the restriction enzyme enzymatic treatment is 33-38 DEG C of processing 12 hours or more;More preferably, the enzyme-activity unit is 10U, and the restriction enzyme enzymatic treatment is 37 DEG C and handles 12 hours.Make limit The method of property restriction endonuclease BseGI inactivations processed is that this field is conventional, preferably, described makes restriction enzyme inactivation be 60-68 DEG C processing loop-stem structure solution 15-25 minutes;More preferably, described to make restriction enzyme inactivation for 65 DEG C of processing loop-stem structures Solution 20 minutes.
This field routine is recovered as described in step (4), preferably electrophoresis recycles;More preferably, the electrophoresis is non-change Property gel electrophoresis, most preferably, the native gel electrophoresis be 8% non-denaturing polyacrylamide gel (PAGE) electrophoresis.
Technical proposal that the invention solves the above-mentioned problems second is that:A kind of gene editing based on restriction endonuclease, targeting positioning And/or regulation and control method comprising:By for express the plasmids of Argonaute intra-system handoff zymoproteins with by above-mentioned preparation side In the ends 5' phosphorylation single stranded DNA cotransfection made from method to the cell of wanted gene editing, targeting positioning and/or regulation and control, into Row gene editing, targeting positioning and/or control manipulation;Preferably, the restriction endonuclease is NgAgo.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention Apply example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) step is simple, of low cost:DNA loop-stem structures design containing restriction enzyme site is simple, without synthesizing double-strand DNA can be obtained using conventional single-stranded primer Composite service with loop-stem structure in two sections of addition fixed sequence programs of aim sequence Precursor;Meanwhile the product of cutting is also DNA single-stranded, without being obtained by denatured double stranded or asymmetric pcr;
(2) phosphorylation is efficient:Shear efficiency is equal to the efficiency of phosphorylation, the 5' of the DNA after restriction enzyme shearing End necessarily carries phosphate group, therefore can prepare the single stranded DNA of 100% phosphorylation;
(3) it is easy to characterize and purifies:Treated that product can be verified and be divided with gel electrophoresis for restriction enzyme From;
(4) applied widely:To target DNA sequence without particular/special requirement, the limitation of unrestricted endonuclease recognition sequence, It can be used for the phosphorylation of substantially any sequence;
(5) it repeats and utilizes, energy conservation and environmental protection:Product after shearing can be recycled by gel electrophoresis, easy to operate, be not necessarily to Special chemical synthesis equipment is easy to be prepared on a large scale 5'-P ssDNA;
(6) application prospect is wide:5'-P ssDNA can be used as gDNA to be used for gene editing system, provide guiding gene targeting The 5'-P ssDNA of the high-purity of shearing, can significantly improve gene editing efficiency.Base can also be significantly improved using the technology It is positioned in the gene target of gene restriction endonuclease, the efficiency of gene regulation, therefore the technology has huge application prospect.
Description of the drawings
Fig. 1 prepares the signal of the single stranded DNA (5'P-ssDNA) of 5 ' end phosphate modifications by restriction enzyme shearing Figure.
Fig. 2 is the preparation method and product detection of 5'P-ssDNA, wherein (a) is to prepare 5'P-ssDNA's using enzyme cutting method Principle schematic, overstriking font base are purpose sequence, are the identification sequence of restriction enzyme BseGI with underscore base, Triangle represents restriction enzyme site;(b) it is that (DNA marker are 20bp DNA for polyacrylamide gel electrophoresis (PAGE) analysis Ladder it) shows;(c) it is sheared for the ssDNA (left side line, relative molecular mass 7485.7) and BseGI of non-phosphorylation modification The Mass Spectrometer Method result of the DNA (right-hand line, relative molecular mass 7565.5) recycled afterwards.
Fig. 3 is that the gDNA (EGFP-gDNA) for EGFP plasmids prepared with enzyme cutting method is sheared for NgAgo system genes The schematic diagram of experiment.
Fig. 4 is effect detections of the gDNA (EGFP-gDNA) in mammalian cell, wherein (a) is EGFP-gDNA and use Position view of the PCR primer of situation on pEGFP-C1 plasmids is sheared in analysis plasmid;(b) each to be used in experiment DNA and its sequence;(c) it is experiment for comparing target gene shear effect and control group;(d) be using fluorescence microscope at As the expression of the EGFP of observation each group sample treatment cell (white scale line length is 1mm);(e) it is each group sample treatment Cellular genome PCR amplification result afterwards.
Specific implementation mode
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient Product specification selects.
Embodiment 1 prepares the single stranded DNA (5'P-ss DNA) of 5' phosphorylations and is characterized to it
5 ' end phosphates are prepared by restriction enzyme BseGI (being purchased from ThermoFisher Scientific) shearing (wherein, thickened portion is recognition site to the process of the single stranded DNA of modification as shown in Figure 1;The direction position of two triangular arrowheads is Restriction enzyme site).
1. loop-stem structure precursor design:The present embodiment is with gDNA (EGFP-gDNA, the sequence for EGFP gene code area For:5'-GTGGTCGGGGTAGCGGCTGAAGCA-3'(SEQ ID No.1) it is purpose DNA, increase at its both ends and contains BseGI The complementary series of endonuclease recognition sequence (GGATGNN^, N represent arbitrary base, and ^ represents shearing site), obtained full sequence For:5'-GCGCGGATGTG GTGGTCGGGGTAGCGGCTGAAGCACATCCGCGC-3'(is see shown in SEQ ID No.2) (italicized item is purpose DNA sequence dna, and underscore part is that BseGI identifies sequence, sees Fig. 2 (a)).By being commercialized primer synthesis Service obtains the sequence ss of high performance liquid chromatography (HPLC) after purification.
2. prepared by loop-stem structure precursor:The sequence with restriction enzyme site that step 1 obtains is dissolved in buffer solution (MgCl22 5mM, Tris-HCl 10mM, pH 8.0;10 μM of DNA final concentrations) (being provided by BseGI supplier products specifications) And (95 DEG C of thermal cycler is handled 5 minutes, is then cooled to 4 degrees Celsius rapidly, and kept for 10 minutes) is made annealing treatment, it obtains Loop-stem structure precursor (shown in such as Fig. 2 (a)).
3. restriction enzyme enzymatic treatment:2 μ L 20000U/mL BseGI are added in the loop-stem structure solution that step 2 obtains (BtsCI) restriction endonuclease, 50 DEG C are handled 12 hours, and being subsequently placed in 80 DEG C of processing makes restriction enzyme inactivate in 20 minutes.
4. digestion products pass through 8% native polyacrylamide gel electrophoresis (native PAGE, Bio-Rad vertical electrophoresis System, 100V voltages 1 hour) verification (shown in result such as Fig. 2 (b)), as a result show:Without restriction enzyme BseGI processing Loop-stem structure precursor relative to 20bp marker bands rearward position be in single band;It is small by BseGI processing 12 Shi Hou, product band is slightly forward relative to the marker bands of 20bp, meets the expection mobility of target product.Glue is utilized later QIAquick Gel Extraction Kit (TAKARA#9762) is tapped and recovered, and carrying out OD260 tests using ultraviolet specrophotometer quantifies, in -20 It DEG C saves backup.
5. EGFP-gDNA (5'-P ssDNA) prepared by Mass Spectrometer Method enzyme cutting method is mutually homotactic with non-phosphorylation modification DNA verifies the phosphorylation efficiency of this method.As shown in Fig. 2 (c), the results showed that DNA is relative to non-phosphorylation after BseGI shearings SsDNA increase the quality (PO of a phosphate group4Matrix amount deducts substituted OH matrix amount, and molecular weight is about 79);Together When, according to mass spectrographic intensity distribution, without apparent miscellaneous peak, illustrate the gDNA yields of correct phosphorylation close to 100%.
Conclusion:By the result of Fig. 2 (c) it is found that the enzyme cutting method described using the present invention can obtain the ends 5' almost 100% The EGFP-gDNA of phosphorylation modification.
GDNA prepared by 2 enzyme cutting method of embodiment guides NgAgo systems to mammalian cell HEK293 (Unite States Standard cultures Object collecting center ATCC) in plasmid shearing
1. by NLS-NgAgo-GK plasmids (for expressing NgAgo endonuclease proteins, Addgene purchases, number:78253) 200ng, pEGFP-C1 plasmid (as the target plasmid for being targeted cutting, express EGFP fluorescins, are purchased from ancient cooking vessel state biology, compile Number:MCV046) 40ng with cationic liposomal transfection reagent passed through by EGFP-gDNA 200ng threes prepared by embodiment one Lipofectamine2000 (Thermo, USA) cotransfection enters HEK293 cells.Cell culture in 24 orifice plates, culture medium be containing The DMEM culture mediums (being purchased from Gibco) of 10% fetal calf serum (being purchased from Gibco), spread about 1 × 10 per hole before testing5A cell culture Overnight.Specifically transfection conditions are:With 25 μ L Opti-MEM culture mediums (being purchased from Gibco) dilution, 1 μ L Lipofectaming 2000,5 minutes are placed at room temperature for after mixing;Another 25 μ L Opti-MEM culture mediums (being purchased from Gibco) three kinds of matter to be transfected of dilution Grain;Above two composite solution is uniformly mixed, is incubated at room temperature 5 minutes, then the DMEM culture mediums of itself and 950 μ L serum-frees are mixed It is even and be added in the culture plate of cell to be transfected;At 37 DEG C, containing 5%CO2Incubator in continue culture 4 hours after, replace Fresh DMEM medium (100 μ g/mL ampicillins of addition) containing 10% fetal calf serum (being purchased from Gibco) continues to cultivate. Principle such as Fig. 3:EGFP-gDNA prepared by embodiment 1, NLS-NgAgo-GK plasmid, pEGFP-C1 plasmids are (as to be sheared Target expresses the EGFP albumen of fluoresced green) cotransfection is to HEK293 cells, the bootable NgAgo albumen pair of EGFP-gDNA PEGFP-C1 plasmids carry out targeting shearing, lower expression of the EGFP in cell.The PCR primer of situation is sheared for analyzing plasmid In the position of pEGFP-C1 shown in such as Fig. 4 (a), shown in primer sequence such as Fig. 4 (b).
2. setting up multiple control groups (shown in such as Fig. 4 (c)):Control I only transfects pEGFP-C1 targeting plasmids (blank control); Compare II transfection NLS-NgAgo-GK expression plasmids, pEGFP-C1 plasmids and non-targeted gDNA (NC-gDNA);Compare III transfections PEGFP-C1 plasmids and EGFP-gDNA (enzyme cutting method preparation), but NgAgo plasmids are not transfected;IV~VI is experimental group, is transfected simultaneously NLS-NgAgo-GK expression plasmids, pEGFP-C1 target plasmids and EGFP-gDNA obtain for more different Phosphorylations GDNA, control IV use be primer Composite service quotient (Shanghai Jie Li Bioisystech Co., Ltd) phosphorylation modification gDNA, V Group is the gDNA that T4 phosphorylases are handled.Processing method is:According to supplier's operation instructions, every 10 μ g wait for phosphorylation T4 polynueleotide kinases (being purchased from NEB) 1 μ L, 5 μ L of T4 ligase buffer solutions (containing ATP) is added in single stranded DNA;Additional addition ATP(25mM)1μL;Deionized water is supplemented to 50 μ L of final volume.It is incubated 12 hours progress phosphorylation reactions for 37 DEG C after mixing, then 65 DEG C are incubated inactivation in 20 minutes, and DNA is finally diluted to 100ng/ μ L with water.VI groups are prepared by enzyme cutting method described in the invention GDNA, in list "+" indicate transfection, "-" indicate untransfected.
3. transfection for the first time supplements transfection 200ng EGFP-gDNA by Lipofectamine2000 respectively after 12 hours.
4. after second of transfection 36 hours, pass through the expression (Fig. 4 (d)) of the intracellular EGFP of fluorescence microscope.
5. in order to avoid because the reduction of pEGFP-C1 transfection efficiencies leads to the possibility of false positive, the gene of each group cell is extracted Group DNA, and utilize the primer pair (Fig. 4 (a), forward primer 1+ reverse primer) and list of EGFP-gDNA targeting restriction enzyme sites both sides The primer pair (forward primer 2+ reverse primers) of side carries out PCR amplification verification to it.The amplification of " forward primer 2+ reverse primers " As a result the transfection efficiency of pEGFP-C1 can be embodied, and the amplification of " forward primer 1+ reverse primers " can embody EGFP bases The shear efficiency of cause.Wherein, 1 sequence of forward primer is:5'-GCCACCATGGTGAGCAAGGGCGAGGAGC-3'(SEQ ID No.3), 2 sequence of forward primer is:5'-CGACTTCTTCAAGTCCGCC-3'(SEQ ID No.4), reverse primer sequences are: TTATCTAGATCCGGTGGATCCC(SEQ ID No.5)。
Conclusion:From the point of view of fluorescence imaging (Fig. 4 (d)), the gDNA (VI groups) for the enzyme cutting method acquisition that the present invention describes effectively drops Low fluorescence intensity into the cell.PCR amplification result (Fig. 4 (e)) is also shown, and the gDNA that enzyme cutting method obtains carries out EGFP plasmids Effective cutting, causes the primer of restriction enzyme site both sides can not to obtain amplified production.In contrast, (IV groups, draw other methods The chemical modification that object Composite service quotient provides;V groups, the phosphorylation modification of T4 phosphorylases) obtain gDNA do not obtain effectively Control of Fluorescence and plasmid shear effect.The above results illustrate that 5'-P gDNA prepared by the enzyme cutting method described through the invention can Efficiently to mediate NgAgo gene editings system in mammalian cell internal cutting EGFP plasmids, EGFP gene expression is caused to be lowered.
Above-described, only presently preferred embodiments of the present invention is not limited to the scope of the present invention, of the invention is upper Stating embodiment can also make a variety of changes.Made by i.e. every claims applied according to the present invention and description Simply, equivalence changes fall within the claims of patent of the present invention.The not detailed description of the present invention is conventional skill Art content.
<110>Shanghai Inst. of Applied Physics Chinese Academy of Sciences
<120>A kind of preparation method and applications of the ends 5' phosphorylation single stranded DNA
<130> P1611448C
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223> EGFP-gDNA
<400> 1
gtggtcgggg tagcggctga agca 24
<210> 2
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<223>EGFP-gDNA containing BseGI endonuclease recognition sequences
<400> 2
gcgcggatgt ggtggtcggg gtagcggctg aagcacatcc gcgc 44
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer 1
<400> 3
gccaccatgg tgagcaaggg cgaggagc 28
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Forward primer 2
<400> 4
cgacttcttc aagtccgcc 19
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Reverse primer
<400> 5
ttatctagat ccggtggatc cc 22

Claims (10)

1. a kind of preparation method of the ends 5' phosphorylation single stranded DNA comprising following steps:
(1) it is inserted into the restriction enzyme site sequence of restriction enzyme at target DNA both ends, forms loop-stem structure precursor, wherein described Target DNA is located at the cyclic single strand region of loop-stem structure precursor, and the restriction enzyme site sequence is located at the stem of loop-stem structure precursor Double-stranded region;
(2) the loop-stem structure precursor that step (1) obtains is dissolved in buffer solution and is made annealing treatment, it is molten to obtain loop-stem structure Liquid;
(3) restriction enzyme enzymatic treatment is added in the loop-stem structure solution for obtaining step (2), so that restriction enzyme is lost after processing It is living, obtain digestion products;
(4) by digestion products recycling obtained by step (3) to get.
2. preparation method as described in claim 1, which is characterized in that the restriction enzyme site sequence contains restriction enzyme Identify that sequence and restriction enzyme site, the restriction enzyme site can be detached with identification sequence;Preferably, the enzyme of the restriction enzyme Enzyme site is located at identification sequence downstream, i.e., the described restriction enzyme site sequence and the base of adjacent target DNA have following sequence:
5’-S1N1N2N3-3’
3’-S2N4N5N6-5’;
The S1For the positive sequence of the identification sequence of restriction enzyme, the S2For the identification sequence of restriction enzyme Reverse sequence, the N1、N2For the reverse complementary sequence of 3 ' two bases in end of purpose DNA, N3-N6For the base of purpose DNA, The restriction enzyme site is located at N2And N3Between and S2And N4Between.
3. preparation method as claimed in claim 2, which is characterized in that the restriction enzyme site sequence also contains protection base sequence Row, wherein S15 ' end have protection base sequence 1, S23 ' end have protection base sequence 2, protection base sequence 1 with protection 2 reverse complemental of base sequence;Preferably, the protection base sequence is the high sequence of the G/C content containing 3-6 base;More Goodly, the protection base sequence 1 is:GCGC, GGGC or CCCG.
4. preparation method as claimed in claim 3, which is characterized in that restriction enzyme described in step (1) be BseGI, BstF5I or BtsCI enzymes, the S1Sequence is GGATG, S2Sequence is CCTAC;
Alternatively, the restriction enzyme is Bts α I or BtsI enzymes, the S1Sequence is GCAGTG, S2Sequence is CGTCAC;
Alternatively, the restriction enzyme is BtsIMutI enzymes, the S1Sequence is CAGTG, S2Sequence is GTCAC;
Alternatively, the restriction enzyme is Bse3DI, BseMI or BsrDI enzyme, the S1Sequence is GCAATG, S2Sequence For CGTTAC.
5. preparation method as claimed in claim 4, which is characterized in that the restriction enzyme described in step (1) is BseGI, Loop-stem structure precursor sequence is:5'-GCGCGGATG+N1N2+ target DNA+CATCCGCGC-3';
And/or the buffer solution described in step (2) includes following component:MgCl210 μM of 5mM, Tris-HCl 10mM, DNA;pH 8.0。
6. preparation method as claimed in claim 5, which is characterized in that the annealing described in step (2) is:Thermal cycler 95 DEG C processing 5 minutes, is then cooled to rapidly 4 DEG C, and kept for 10 minutes;
And/or the restriction enzyme enzymatic treatment described in step (3) is 33-38 DEG C of processing 12 hours or more, it is described it is restricted in The enzyme-activity unit of enzyme cutting is 8-15U;Preferably 37 DEG C are handled 12 hours, and the enzyme-activity unit of the restriction enzyme is 10U.
7. such as preparation method described in claim 5 or 6, which is characterized in that so that restriction enzyme is inactivated described in step (3) Loop-stem structure solution is handled for 60-68 DEG C 15-25 minutes;Preferably 65 DEG C handle loop-stem structure solution 20 minutes.
8. preparation method as described in claim 1, which is characterized in that be recovered as electrophoresis recycling described in step (4);Preferably Ground, the electrophoresis are native gel electrophoresis;More preferably, the native gel electrophoresis is solidifying for 8% non-denaturing polyacrylamide Gel electrophoresis.
9. a kind of gene editing, targeting positioning and/or regulation and control method based on restriction endonuclease comprising:It will be used to express The plasmid of Argonaute intra-system handoff zymoproteins and pass through the ends 5' phosphorylation made from any one of claim 1-8 preparation methods Single stranded DNA cotransfection to wanted gene editing, targeting positioning and/or regulating cell in, carry out gene editing, targeting positioning And/or regulation and control processing.
10. the gene editing based on restriction endonuclease, targeting positioning and/or regulation and control method, feature exist as claimed in claim 9 In the restriction endonuclease is NgAgo.
CN201710067173.9A 2017-02-06 2017-02-06 A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA Pending CN108396043A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710067173.9A CN108396043A (en) 2017-02-06 2017-02-06 A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710067173.9A CN108396043A (en) 2017-02-06 2017-02-06 A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA

Publications (1)

Publication Number Publication Date
CN108396043A true CN108396043A (en) 2018-08-14

Family

ID=63094437

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710067173.9A Pending CN108396043A (en) 2017-02-06 2017-02-06 A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA

Country Status (1)

Country Link
CN (1) CN108396043A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480568B (en) * 2020-12-30 2024-04-26 安诺优达基因科技(北京)有限公司 Detection method and kit for phosphorylation reaction

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054299A (en) * 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
CN1399679A (en) * 1999-10-04 2003-02-26 西托吉尼克斯公司 Altering gene expression with ssDNA produced in vivo

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054299A (en) * 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
CN1399679A (en) * 1999-10-04 2003-02-26 西托吉尼克斯公司 Altering gene expression with ssDNA produced in vivo

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALFRED PINGOUD等: "Structure and function of type II restriction endonucleases", 《NUCLEIC ACIDS RESEARCH》 *
李峰主编: "《分子生物学实验》", 31 December 2015, 华中科技大学出版社 *
杨洁等: "新一代基因组编辑技术-NgAgo-gDNA", 《生物学教学》 *
诸兵: "自组装DNA纳米材料结构与功能的研究", 《中国博士学位论文全文数据库基础科学辑》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480568B (en) * 2020-12-30 2024-04-26 安诺优达基因科技(北京)有限公司 Detection method and kit for phosphorylation reaction

Similar Documents

Publication Publication Date Title
US11802277B2 (en) Thermostable Cas9 nucleases
CN108642055B (en) sgRNA capable of effectively editing pig miR-17-92 gene cluster
US9879283B2 (en) CRISPR oligonucleotides and gene editing
Oh et al. Design, assembly, production, and transfection of synthetic modified mRNA
CN109136248B (en) Multi-target editing vector and construction method and application thereof
CN106957858A (en) A kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly
CN110699407B (en) Preparation method of long single-stranded DNA
KR20230021657A (en) Enzymes containing RUVC domains
BR112020025319A2 (en) COMPOSITIONS, SYSTEMS AND METHODS OF AMPLIFICATION BASED ON CRISPR / CAS AND TRANSPOSASE
Klanert et al. Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells
Voß et al. Production of supercoiled multimeric plasmid DNA for biopharmaceutical application
CN107488655B (en) Method for removing 5 &#39;and 3&#39; adaptor connection by-products in sequencing library construction
CN108396043A (en) A kind of preparation method and applications of the ends 5` phosphorylation single stranded DNA
US8486666B2 (en) Removal of the guanine cap on the 5′ terminus of RNA
CN113728097A (en) Enzymes with RUVC domains
CN110499334A (en) CRISPR/SlugCas9 gene editing system and its application
CN116004716A (en) Method for efficiently editing genes by using replication dCAS9-FokI system
AU2022335499A1 (en) Enzymes with ruvc domains
CN113817778A (en) Method for enhancing mRNA stable expression by nucleolin
CN114990093A (en) Protein sequence MINI RFX-CAS13D with small amino acid sequence
CN112266911B (en) Nucleic acid molecules
CN114592022A (en) Long-chain TNA synthesis method based on DNA template
Wu et al. Ribonucleoproteins mediated efficient in vivo gene editing in skin stem cells
Tonooka et al. One-base excess adaptor ligation method for walking uncloned genomic DNA
CN109402153B (en) Construction of protein label vector and expression and detection method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20180814

WD01 Invention patent application deemed withdrawn after publication