CN119144630A - Non-replicative recombinant phage packaging plasmid and application thereof - Google Patents
Non-replicative recombinant phage packaging plasmid and application thereof Download PDFInfo
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Abstract
The invention provides a non-replicative recombinant phage packaging plasmid and application thereof, in particular to a non-replicative recombinant phage packaging plasmid and a method for preparing ssDNA and mcDNA, wherein the plasmid comprises an initial sequence from a phage replication origin, a target sequence and a termination sequence from the phage replication origin, the initial sequence at least comprises a sequence shown as x-v position in SEQ ID NO:1 from 5 'to 3', the termination sequence at least comprises a sequence shown as 1-y position and z-w position in SEQ ID NO:2, and x is not less than 291,329 and not more than v is not more than 381,67 and y is not less than z is not more than 279,312 and not more than w is not more than 330. Compared with the original full-length sequence, the non-replicating recombinant phage packaging plasmid has at least one segment of deletion of the initial sequence and/or the termination sequence of the replication origin, and can still generate circular single-stranded DNA containing the target sequence and the deleted phage replication origin sequence, thereby providing safer single-stranded DNA donors containing fewer non-target sequences for downstream applications such as gene editing, ssDNA and mcDNA preparation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a non-replicative recombinant phage packaging plasmid and an application method for preparing ssDNA and mcDNA based on the non-replicative recombinant phage.
Background
Single-stranded DNA is considered as a biological material, has great application potential in many biological reactions and has wide application in DNA nanotechnology, however, synthesis of long single-stranded DNA is difficult to ensure yield, yield and satisfactory cost performance due to the limitation of chemical synthesis methods, so that the preparation methods of long single-stranded DNA which are commonly used at present mainly include reverse transcription methods, enzymatic degradation methods, denaturing High Performance Liquid Chromatography (HPLC) methods, magnetic bead Biotin (Biotin) modification methods, asymmetric PCR methods, RCA methods and the like, require the in vivo or in vitro action of biological enzymes and some auxiliary denaturation means for long single-stranded DNA. However, in practical applications, these methods have problems such as low yield and high cost.
The helper phage method is a newer method for preparing single-stranded DNA. The basic principle is to construct a plasmid containing an M13 origin of replication (M13 ori) or an F1 origin of replication (F1 ori), transfer it into a host cell containing an F factor and then to infect it with a defective helper phage. Helper phages can help the plasmid form single stranded DNA and wrap into phages that secrete out of the host cell. The method has low cost and high yield, but the formed single-stranded DNA contains a necessary M13ori/f1ori conserved sequence of 2-3 nt.
It has been reported in the literature that pUC18 can be converted into a recombinant phage packaging plasmid for producing custom single-stranded DNA by adding four modules, wild-type M13 ori for the start sequence (M13 ori start), cleavage site, M13 PS for phage particle export, and mutant M13 ori as the termination sequence (M13 ori terminator), and the finally produced single-stranded circular DNA still has 381 bases of wild-type M13 ori conserved sequence （Nafisi, Parsa M.; Aksel, Tural; Douglas, Shawn M. Synthetic Biology (Oxford, United Kingdom) (2018), 3(1), ysy015）. but how to further shorten the length of the conserved sequence is still a problem to be solved.
The documents referred to above are incorporated herein in their entirety.
Disclosure of Invention
The inventor finds that after a certain deletion of a starting sequence (M13 ori start) and a termination sequence (M13 ori terminator) on a recombinant phage packaging plasmid, circular single-stranded DNA containing a target sequence and a phage replication origin (Origin of replication, ori) sequence can be generated, so that safer single-stranded DNA donors containing fewer non-target sequences can be provided for downstream applications such as gene editing, micro-circular DNA (MINICIRCLE DNA, MCDNA) preparation and the like, the target sequence length can reach thousands nt, the conserved sequence of the finally obtained circular single-stranded DNA is shorter than that of a traditional full-length phage replication origin sequence, and the potential influence of the phage replication origin serving as a framework sequence on the target sequence can be reduced. Specifically, the present invention includes the following:
In one aspect, the invention provides a recombinant phage packaging plasmid comprising a start sequence (M13 ori start), a target sequence (GOI) and a stop sequence (M13 ori terminator), wherein the start sequence comprises at least the sequence shown as x-v position in SEQ ID NO:1, the stop sequence comprises at least the sequence shown as 1-y position and z-w position in SEQ ID NO:2, wherein x is not less than 291,329 and not more than 381,67 and y is not less than 279,312 and not more than w is not less than 330, x and y, z, v, w are both positive integers, the start sequence is not SEQ ID NO:1 and/or the stop sequence is not SEQ ID NO:2. The sequence shown in SEQ ID NO. 1 is a wild type M13 ori sequence, the sequence shown in 381 nt,SEQ ID NO:2 is a mutant type M13 ori sequence, the length is 330 nt, and the 1-319 positions of the sequence shown in SEQ ID NO. 2 are consistent with the 1-319 positions of the sequence shown in SEQ ID NO. 1. The invention provides a starting sequence and a stopping sequence, wherein at least one sequence is inconsistent with SEQ ID NO. 1 or SEQ ID NO. 2, namely at least one sequence has deletion compared with the full-length starting sequence or stopping sequence, and circular single-stranded DNA containing a target sequence can be generated.
In some embodiments, the recombinant phage packaging plasmid has a deletion in the start and/or stop site, and the resulting circular single stranded DNA containing the target sequence has a deletion in the M13 ori sequence compared to the sequence shown in wild type SEQ ID NO. 1. In some embodiments, the recombinant phage packaging plasmid has a deletion in the start and/or stop site, and the resulting circular single stranded DNA containing the target sequence has an M13 ori sequence that is identical to the sequence shown in wild type SEQ ID NO. 1, and NO deletion.
Unless otherwise indicated, integers referred to herein are positive integers greater than 0. Unless otherwise indicated, reference herein to a nucleic acid sequence is in a 5 'to 3' direction from left to right.
In some embodiments, x is an integer less than 292, in some embodiments, x is an integer less than 290, in some embodiments, x is an integer less than 200, in some embodiments, x is an integer less than 190, in some embodiments, x is an integer less than 180, in some embodiments, x is an integer less than 260, in some embodiments, x is an integer less than 250, in some embodiments, x is an integer less than 240, in some embodiments, x is an integer less than 230, in some embodiments, x is an integer less than 220, in some embodiments, x is an integer less than 210, in some embodiments, x is an integer less than 200, in some embodiments, x is an integer less than 170, in some embodiments, x is an integer less than 160, in some embodiments, x is an integer less than 150, in some embodiments, x is an integer less than 140, in some embodiments, x is an integer less than 40, in some embodiments, x is an integer less than 80, in some embodiments, x is an integer less than 40, in some embodiments, x is an integer less than 100, in some embodiments, x is an integer less than 80, in some embodiments, x is an integer less than 100, in some embodiments, in some x is an integer less than 100, x is an integer less than 10.
In some embodiments, the starting sequence of the recombinant phage packaging plasmid is the sequence shown in SEQ ID NO. 1 at position x-329, wherein x is 1、2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290 or 291, in order from 5 'to 3'.
In some embodiments, the starting sequence of the recombinant phage packaging plasmid is the sequence shown in position x-381 of SEQ ID NO.1, in order from 5 'to 3'.
In some embodiments, the starting sequence is the sequence set forth in SEQ ID NO. 1. In some embodiments, the starting sequence is the sequence set forth in SEQ ID NO. 5 or SEQ ID NO. 19.
In some embodiments, the recombinant phage packaging plasmid comprises a start sequence comprising at least the sequence shown at positions 291-381 of SEQ ID NO.1, a target sequence comprising at least the sequences shown at positions 1-67 and 279-312 of SEQ ID NO.2, and a stop sequence, in order from 5 'to 3'.
In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-y and z-w in SEQ ID NO. 2, in order from 5 'to 3'. In some embodiments, y is an integer greater than 66, in some embodiments, y is an integer greater than 70, in some embodiments, y is an integer greater than 80, in some embodiments, y is an integer greater than 90, in some embodiments, y is an integer greater than 100, in some embodiments, y is an integer greater than 110, in some embodiments, y is an integer greater than 120, in some embodiments, y is an integer greater than 130, in some embodiments, y is an integer greater than 140, in some embodiments, y is an integer greater than 150, in some embodiments, y is an integer greater than 160, in some embodiments, y is an integer greater than 170, in some embodiments, y is an integer greater than 180, in some embodiments, y is an integer greater than 190, in some embodiments, y is an integer greater than 200, in some embodiments, y is an integer greater than 210, in some embodiments, y is an integer greater than 220, y is an integer greater than 230, in some embodiments, y is an integer greater than 250.
In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown as positions 1-y and z-w in SEQ ID NO. 2, wherein y is 67, 68, 69, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300, and z is 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 278, in order from 5 'to 3'.
In some embodiments, w is 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, or 330.
In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences as set forth in positions 1-67 and 279-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-70 and 260-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences as set forth in positions 1-80 and 250-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-90 and 240-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-100 and 230-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-110 and 220-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-120 and 210-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-130 and 200-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-140 and 190-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-150 and 180-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-160 and 170-312 of SEQ ID NO. 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown as positions 1-150 and 161-312 of SEQ ID NO. 2.
In some embodiments, the termination sequence is selected from one of the sequences shown in SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:21.
Further, the recombinant phage packaging plasmid further comprises at least one deoxyribose or restriction enzyme cleavage site. In some embodiments, to obtain linear single stranded DNA, a deoxyribozyme cleavage site or a restriction endonuclease cleavage site is also provided. In some embodiments, the deoxyribose or restriction enzyme cleavage site is 1. In some embodiments, the deoxyribose or restriction enzyme cleavage sites are 2 and are disposed at both ends of the target sequence, respectively.
In another aspect, the invention also provides a method for preparing phage circular single-stranded DNA, comprising designing and constructing recombinant phage packaging plasmid, wherein the recombinant phage packaging plasmid is any one of the recombinant phage packaging plasmids, transforming host cells, specifically competent cells, with the recombinant phage packaging plasmid and auxiliary plasmid, and carrying out replication and extraction to obtain phage circular single-stranded DNA.
In another aspect, the invention also provides a phage circular single-stranded DNA comprising a target sequence and a deleted M13 ori sequence, the deleted M13 ori sequence comprising at least the sequence shown as 1-y and z-329 bits in SEQ ID NO. 1, wherein 67.ltoreq.y < z.ltoreq.279, and z is an integer at least 2 greater than y, in some embodiments z is an integer at least 6 greater than y, in some embodiments z is an integer at least 11 greater than y, in some embodiments z is an integer at least 21 greater than y, in some embodiments z is an integer at least 31 greater than y, in some embodiments z is an integer at least 41 greater than y, in some embodiments z is an integer at least 51 greater than y, in some embodiments z is an integer at least 61 greater than y, in some embodiments z is an integer at least 81 greater than y, in some embodiments z is an integer at least 91 greater than y, in some embodiments z is an integer at least 101 greater than y, in some embodiments is an integer greater than y is at least 150, in some embodiments z is greater than y is an integer greater than y is at least 150. In some embodiments, the deleted M13 ori sequence comprises at least the sequences shown as positions 1-67 and 279-381 of SEQ ID NO. 1, and is not the sequence shown as SEQ ID NO. 1.
Preparation of circular Single-stranded DNA referring to FIG. 1, wild-type M13 ori is a sequence of full-length 381 nt as shown in SEQ ID NO. 1. When the starting sequence of the recombinant phage packaging plasmid adopts the full-length sequence (SEQ ID NO: 1) of the wild type M13 ori, the stopping sequence adopts the full-length sequence (SEQ ID NO: 2) of the mutant type M13 ori, and the recombinant phage packaging plasmid is combined with the circular single-stranded DNA generated by the auxiliary plasmid, wherein M13 ori is the full-length sequence of the wild type M13 ori. When the recombinant phage packaging plasmid has its initial sequence providing the x-v position of the full length sequence of wild type M13 ori shown in SEQ ID NO. 1 and its final sequence providing the 1-y position and z-w position of the full length sequence of mutant type M13 ori shown in SEQ ID NO. 2, there must be a deletion in the middle section of the final sequence because 67.ltoreq.y < z.ltoreq.279 and z is an integer at least 2 larger than y. In some embodiments, when x is also an integer at least 2 greater than y, the part of the termination sequence deleted cannot be fully complemented by the initial sequence, so that the packaged circular single-stranded DNA has a certain deletion of the M13 ori sequence compared with the wild type full-length sequence shown in SEQ ID NO. 1, when 329 is less than or equal to v <381, the end of the circular single-stranded DNA can be deleted by two sections of sequences, namely, the sequence between y and z or x and the sequence shown in the section z to 381 respectively, when x is less than or equal to y, the part of the termination sequence deleted can be fully complemented by the initial sequence, so that the packaged circular single-stranded DNA has NO deletion of the section.
For ease of understanding, the present invention provides a method for preparing circular single-stranded DNA shown in FIG. 1, but is not limited thereto. Any circular single stranded DNA, when M13 ori is M13 ori comprising at least deletions of the 1 st to 67 th and 279 to 381 th sequences as shown in SEQ ID NO:1, is within the scope of the present invention.
In comparison to the full length fixed sequence shown in SEQ ID NO.1, in embodiments, the fixed sequence lacks 10 consecutive bases, in embodiments, the fixed sequence lacks 20 consecutive bases, in embodiments, the fixed sequence lacks 30 consecutive bases, in embodiments, the fixed sequence lacks 40 consecutive bases, in embodiments, the fixed sequence lacks 50 consecutive bases, in embodiments, the fixed sequence lacks 60 consecutive bases, in embodiments, the fixed sequence lacks 70 consecutive bases, in embodiments, the fixed sequence lacks 80 consecutive bases, in embodiments, the fixed sequence lacks 90 consecutive bases, in embodiments, the fixed sequence lacks 120 consecutive bases, in embodiments, the fixed sequence lacks 130 consecutive bases, in embodiments, the fixed sequence lacks 60 consecutive bases, in embodiments, the fixed sequence lacks 140 consecutive bases, in embodiments, the fixed sequence lacks 150 consecutive bases, in embodiments, the fixed sequence lacks 170 consecutive bases, in embodiments, the fixed sequence lacks 150 consecutive bases, in embodiments, the fixed sequence lacks 100 consecutive bases, in embodiments, and the fixed sequence lacks 120 consecutive bases, in embodiments, in the fixed sequence lacks 170 consecutive bases, in embodiments, in the fixed sequence lacks 150 consecutive bases, in embodiments, in the fixed sequence lacks 170 consecutive bases, in embodiments, in the fixed sequence has been deleted 120 consecutive bases, the fixed sequence lacks 220 consecutive bases, in some embodiments 230 consecutive bases, in some embodiments 240 consecutive bases, and in some embodiments 250 consecutive bases.
Further, the phage circular single-stranded DNA further comprises at least one deoxyribose or restriction enzyme cleavage site. In some embodiments, the phage circular single-stranded DNA comprises a deoxyribose cleavage site or restriction enzyme cleavage site, and linear single-stranded DNA can be obtained by linear cleavage by a deoxyribose or restriction enzyme. In some embodiments, the phage circular single-stranded DNA comprises two deoxyribose or restriction enzyme cleavage sites, one on each side of the target sequence, and linear single-stranded DNA of the target sequence can be obtained by linear cleavage by a deoxyribose or restriction enzyme.
In another aspect, the invention also provides a preparation method of the micro-ring DNA, which comprises the steps of preparing any of the circular single-stranded DNA, and then carrying out in-vitro complementation on the circular single-stranded DNA to finally obtain the micro-ring DNA. In some embodiments, the microcircular DNA is obtained by asymmetric amplification of single stranded DNA.
In some embodiments, one single strand of the micro-circular DNA consists of the sequences shown in SEQ ID NO. 1 at positions 1-67 and 279-329.
The traditional method for preparing the micro-circular DNA is that the parent plasmid (PARENTAL PLASMID, PP) is subjected to site-specific recombination under the action of LR cloning enzyme and is converted into two circular DNA, one of which contains a large amount of bacterial skeleton sequences, called micro-plasmid (MINIPLASMID, MP), and the other is eukaryotic expression frame containing target genes, namely micro-circular DNA (MC). However, the method has the following defects of high price and high cost of the LR cloning enzyme, 2, large loss and small obtaining amount of the micro-ring DNA obtained by gel recovery and purification, 3, complicated steps, and difficult subsequent micro-ring DNA purification and recovery caused by the fact that the micro-carrier is destroyed by restriction enzyme digestion after LR reaction. The method of the micro-ring DNA provided by the invention is simpler and more convenient, and the cost is lower.
The invention provides a recombinant phage packaging plasmid, which comprises a starting sequence, a target sequence and a termination sequence, wherein the starting sequence at least comprises a section of sequence shown as x-v position in SEQ ID NO. 1, the termination sequence at least comprises two sections of sequence shown as 1-y position and z-w position in SEQ ID NO. 2, and x is not less than 291,329 and not more than 381,67, y is not less than z is not less than 279,312 and not more than w is not more than 330. The circular single-stranded DNA prepared by the recombinant phage packaging plasmid provided by the invention has the advantages that the M13 ori sequence is deleted, the length is shorter, and the influence of the M13 ori serving as a framework sequence on a target sequence can be reduced.
Drawings
For a better understanding of the invention and to show more clearly how it may be carried into effect, features according to embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:
FIG. 1 is a schematic diagram of the structure of wild type M13 ori, deleted initiation sequence, deleted termination sequence and deleted M13 ori.
FIG. 2 shows the sequence structure of recombinant phage packaging plasmid and corresponding circular single-stranded DNA, wherein A is pMPdID C-css DNA, B is pMT2d5C9MRdM75-css DNA, C is pMT2d5C9MRdM R16-css DNA, and the broken line part is the deleted sequence.
FIG. 3 shows a detection chart of css-DNA agarose gel electrophoresis. Lane M, standard; lane 1: pMPdID2C-cssDNA, lane 2: pMT2D5C9-cssDNA, (B) Lane M: standard, lane 1: NA, lane 2: NA, lane 3: NA, lane 4: pMT2d5C9M10-cssDNA, lane 5: pMT2D5C9R10-cssDNA, (C) Lane M: standard, lane 1: pMT2D5C9MRd-cssDNA clone 1, lane 2: pMT2D 9MRd-cssDNA clone 2, (D) Lane M: standard, lane 1: NA, lane 2: NA, lane 3: pMT2D 9MRdM75-cssDNA clone 1, lane 4: pMT2D 9 MRdM758-3496 clone 1, lane 2: pMT2D 75 MR9R 125-379 MR2; pMT 2D-379 MR9R 57, and pMT 2: pMT 2D-025 clone 325M 1-325F, and (M) lane 75-325M 9M 75-325F, and (M) lane 125-125).
FIG. 4 pMT2d5C9MRdM75R16dS-cssDNA agarose gel electrophoresis pattern. Lane M is a standard sample, lane 1 is pMT2d5C9MRdM75R16dS-cssDNA, wherein the white horizontal line labeled interval is the predicted site of the target product.
FIG. 5 shows a gel electrophoresis pattern of cleavage products of pMT2d5C9-cssDNA, pMT2d5C9-ssDNA and pMT2d5C 9-ssDNA. Lane M is standard, lane 1 is pMT2d5C9-cssDNA, lane 2 is pMT2d5C9-ssDNA, and Lane 3 is pMT2d5C9-ssDNA exoenzyme digestion identification.
FIG. 6 shows a sequencing map of pMT2d5C9MRdM83R16-cssDNA ori.
FIG. 7 is a drawing showing css-DNA agarose gel electrophoresis detection, wherein the white horizontal line mark interval is the expected site of the target product. The electrophoresis pattern of the packaging product of pMT1D3C2, (B) the electrophoresis pattern of the packaging product of pMT1D5C1, (C) the electrophoresis pattern of the packaging product of pMT2D5C10, (D) the electrophoresis pattern of the packaging product of pMT2D3C1, (E) the electrophoresis pattern of the packaging product of pMT2D5C10, (F) the electrophoresis pattern of the packaging product of pMT2D5C9MRdR, and (G) the electrophoresis pattern of the packaging product of pMT2D5C9MRdM R16.
FIG. 8 shows gel electrophoresis detection patterns of products obtained after digestion and column purification of mcDNA produced after 2, 3 and 4 cycles of circular amplification, respectively, using pMPdID2C-cssDNA as a template.
Detailed Description
Definition of the definition
In order to provide a clear and consistent understanding of the terms used in the description of the present invention, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The use of the word "a" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "an" but it is also known to the meaning of "one or more", "at least one" and "one or more". Similarly, the word "another" may mean at least a second or a plurality.
The word "comprising" (and any form of comprising, such as "comprising" and "comprises"), "having" (and any form of having, "having", "including" and "containing") as used in this specification and claims is inclusive and open-ended and does not exclude additional unrecited elements or process steps.
As used herein, "recombinant phage packaging plasmid" or "Phagemid" are used interchangeably and are a class of artificially constructed, specific types of vectors containing single-stranded phage packaging sequences, replicons, as well as plasmid replicons, cloning sites, marker genes.
As used herein, "micro-circular DNA," "mcDNA," "MINICIRCLE," and "MINICIRCLE DNA" are used interchangeably. The micro-ring DNA is a novel small-ring supercoiled expression frame, lacks a framework sequence of bacteria such as resistance marker genes, and the like, and enhances the safety in clinical application. Compared with virus vector and plasmid vector, the micro-ring DNA reduces the possibility of inflammation and gene silencing, and the expression period is longer, and the gene expression strength is enhanced by 10-1000 times.
Examples the invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed to limit the scope of the invention in any way.
Unless defined otherwise or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Although the present invention has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope of the invention as defined in the claims.
The experimental methods not specifically described in the present invention are carried out according to the methods described in the "molecular cloning Experimental guidelines (Fourth Edition) J.Sam Brooks (Joseph. Sambrook, molecular Cloning: A Laboratory Manual (Fourth Edition)), or according to the specifications of the related products. As used herein, all terms herein are to be understood in their ordinary sense as known in the art unless otherwise indicated. The biological reagents used in the invention are all available from commercial sources without special description, and the brand information of the experimental main materials is shown in the following table 1.
TABLE 1 Main materials
EXAMPLE 1 production of circular single stranded DNA by pMPdID2C
1) Recombinant phage packaging plasmid pMPdID C was obtained by PCR subcloning, consisting of the starting sequence, the target sequence, and the termination sequence in order. The initial sequence is the full-length initial sequence shown as SEQ ID NO. 1, the sequence length is 381 nt, the termination sequence is the full-length termination sequence shown as SEQ ID NO. 2, the sequence length is 330 nt, and the target sequence is shown as SEQ ID NO. 4.
2) Recombinant phage packaging plasmid and helper plasmid transformation
2.1. Competent cells DH 5. Alpha. (Norvezan) were removed from-80℃and rapidly thawed on ice.
2.2. To be transformed 25 ng Phagemid pMPdID2C and 50 ng helper plasmid were added to 100 μl of competent cells, the walls of the flick tube were mixed well and left to stand on ice for 30min.
2.3.42 After heat shock 45 sec in a water bath at C, the mixture is quickly placed on ice for standing 2: 2 min.
2.4. Add +900. Mu.L LB or SOC broth (without antibiotics) to the centrifuge tube, mix well and place at 30℃with 220 rpm shake 1 h.
2.5. 100. Mu.L of the bacterial liquid was uniformly spread on LB solid medium plates containing chloramphenicol and kanamycin antibiotics.
2.6. The plates were inverted and incubated overnight.
2.7. The bacterial lot number and clone number were labeled.
2.8. From the plates, the monoclonals were individually picked into 10 mL of 2 XYT (antibiotic-containing) liquid medium, 30 ℃,270 rpm, and incubated 16-24 h.
3) Phage single-stranded DNA extraction
3.1. Bacterial culture 4000 rcf,4 ℃ 15 min.
3.2. PEG8000 4 g/100 mL, naCl 3 g/100 mL were weighed into a new centrifuge tube and added to the centrifuged supernatant to avoid sedimentation below the agitation. Mix well at room temperature until the solid is completely dissolved.
3.3. Incubate on ice 30 min.
3.4.5000 Rcf,4 ℃, centrifuge 30 min, discard supernatant.
3.5. The pellet was resuspended with 133. Mu.L/TE.
3.6. P2 was added to 2 volumes of plasmid extraction kit and gently mixed.
3.7. P3 was added to 1.5 volumes of plasmid extraction kit and gently mixed.
3.8. Rnase a was added and incubated at room temperature for 10 min ℃ in an ice bath 10 min,16000 rcf,4 ℃ and centrifuged for 30 min.
3.9. The supernatant was aspirated and an equal volume of absolute ethanol pre-chilled at-20 ℃ was added and precipitated overnight at-20 ℃.
3.10.16000 Rcf,4 ℃, centrifuge 30 min, discard supernatant.
3.11. Washing, adding 70% ethanol, 16000 rcf,4 ℃, centrifuging for 30 min, and discarding the supernatant.
3.12. The above steps are repeated.
3.13. Agarose gel electrophoresis and sequencing were performed on the extracted M13 phage genome.
PMPdID2C-cssDNA total length 4968 nt, wherein the target sequence 4587 nt, the generated M13 ori sequence is the wild type M13 ori total length sequence shown in SEQ ID NO. 1, the sequence length is 381 nt, and agarose gel electrophoresis is shown in FIG. 3.
EXAMPLE 2 formation of the cyclic form of pMT2d5C9 Single-stranded DNA and linear single-stranded DNA
1) Circular single stranded DNA
The recombinant phage packaging plasmid pMT2d5C9 is obtained by PCR subcloning and consists of a starting sequence, a ribozyme cleavage sequence, a target sequence and a termination sequence in order. The initial sequence is the initial sequence of deletion shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 lacks the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO. 1 and keeps consistent with 291-381 bit sequence, the termination sequence is the termination of deletion shown as SEQ ID NO. 6, the termination sequence with the length of 312 nt,SEQ ID NO:6 lacks the 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO. 2 and keeps consistent with 1-312 bit sequence, the ribozyme cutting sequence is shown as SEQ ID NO. 3, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
PMT2d5C9-cssDNA total length 5058 nt, wherein ribozyme cleavage sequence 82 nt target sequence 4595 nt, M13 ori is still full-length sequence, as shown in SEQ ID NO. 5, sequence length 381 nt, agarose gel electrophoresis is shown in FIG. 3.
2) Linear single stranded DNA
Further ribozyme cleavage was performed on pMT2d5C9-cssDNA, the reaction system was shown in Table 2, and after preparation, the mixture was thoroughly mixed, and the reaction was carried out by short centrifugation using a mini centrifuge to eliminate air bubbles in the system at 37℃for 30 min.
TABLE 2 cleavage reaction System
And (3) carrying out single-chain verification on the enzyme digestion reaction product, directly hydrolyzing the reaction solution of the digestion reaction system by using exoenzyme Exo V, and then detecting by using gel electrophoresis, wherein the detection result is shown in figure 5. As can be seen from FIG. 5, the cleaved product can be hydrolyzed by an exonuclease, i.e., pMT2d5C9-ssDNA was successfully prepared.
EXAMPLE 3 production of circular single stranded DNA by pMT2d5C9M10
The recombinant phage packaging plasmid pMT2d5C9M10 was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 7, the termination sequence with the length of 302 nt,SEQ ID NO:7 is deleted from the 151-160 bit sequence and 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with 1-150 and 161-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
The total length of pMT2d5C9M10-cssDNA is 49666 nt, wherein the target sequence 4595 nt, the deleted M13 ori is shown in SEQ ID NO. 8, the deletion of 10 nucleotides at 151-160 positions is compared with the wild type M13 ori, the length is 371 nt, and agarose gel electrophoresis is shown in FIG. 3.
EXAMPLE 4 production of circular single stranded DNA by pMT2d5C9R10
The recombinant phage packaging plasmid pMT2d5C9R10 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in sequence. The initial sequence is a deleted initial sequence shown in SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 lacks the 1-290 bit sequence of the full-length initial sequence shown in SEQ ID NO. 1 and keeps consistent with 291-381 bit sequence, the termination sequence is a deleted termination sequence shown in SEQ ID NO. 9, the termination sequence with the length of 302 nt,SEQ ID NO:9 lacks the 253-262 bit sequence and 313-330 bit sequence of the full-length termination sequence shown in SEQ ID NO. 2 and keeps consistent with 1-252 and 263-312 bit sequences, and the target sequence is shown in SEQ ID NO. 4. Other steps refer to example 1.
The total length of pMT2d5C9R10-cssDNA is 49666 nt, wherein the target sequence 4595 nt, the deleted M13 ori is shown as SEQ ID NO. 10, the deletion of 253-262 10 nucleotides compared with the wild type M13 ori, the length is 371 nt, and agarose gel electrophoresis is shown in FIG. 3.
EXAMPLE 5 production of circular single stranded DNA by pMT2d5C9MRd
The recombinant phage packaging plasmid pMT2d5C9MRd was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown in SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted by 1-290 bits of the full-length initial sequence shown in SEQ ID NO.1, the initial sequence is consistent with 291-381 bits of the sequence, the termination sequence is a deleted termination sequence shown in SEQ ID NO. 11, the termination sequence with the length of 200 nt,SEQ ID NO:11 is deleted by 151-262 bits and 313-330 bits of the full-length termination sequence shown in SEQ ID NO. 2, the initial sequence is consistent with 1-150 and 263-312 bits of the sequence, and the target sequence is shown in SEQ ID NO. 4. Other steps refer to example 1.
PMT2d5C9MRd-cssDNA total length 4864 nt, wherein the target sequence 4595 nt, the deleted M13 ori is shown in SEQ ID NO. 12, the deletion of 112 nucleotides at 151-262 compared with the wild type M13 ori, the length 269 nt, agarose gel electrophoresis is shown in FIG. 3.
EXAMPLE 6 production of circular single stranded DNA by pMT2d5C9MRd M75
The recombinant phage packaging plasmid pMT2d5C9MRdM was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO.1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 13, the termination sequence with the length of 125 nt,SEQ ID NO:13 is deleted from the 76-232 bit sequence and 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO.2, and is consistent with 1-75 and 233-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
The total length of pMT2d5C9 MRdM-cssDNA is 4789 nt, the target sequence 4595 nt is shown in SEQ ID NO. 14, the deleted M13 ori is deleted by 187 nucleotides at 76-232 compared with the wild type M13 ori, the length is 194 nt, and agarose gel electrophoresis is shown in FIG. 3.
EXAMPLE 7 production of circular single stranded DNA by pMT2d5C9MRdM75R8
The recombinant phage packaging plasmid pMT2d5C9MRdM R8 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted by 1-290 bits of the full-length initial sequence shown as SEQ ID NO. 1, the initial sequence is consistent with 291-381 bits of the sequence, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 15, the termination sequence with the length of 117 nt,SEQ ID NO:15 is deleted by 76-270 bits and 313-330 bits of the full-length termination sequence shown as SEQ ID NO. 2, the initial sequence is consistent with 1-75 and 271-312 bits of the sequence, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
PMT2d5C9MRdM R8-cssDNA total length 4781 nt, wherein target sequence 4595 nt, deleted M13 ori as shown in SEQ ID NO. 16, deletion of 195 nucleotides at 76-270 compared with wild type M13 ori, length 186 nt, agarose gel electrophoresis as shown in FIG. 3.
EXAMPLE 8 production of circular single stranded DNA by pMT2d5C9MRdM75R16
The recombinant phage packaging plasmid pMT2d5C9MRdM R16 was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 17, the termination sequence with the length of 109 nt,SEQ ID NO:17 is deleted from the 76-278 bit sequence and 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with 1-75 and 279-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
PMT2d5C9MRdM R16-cssDNA total length 4773 nt, wherein target sequence 4595 nt, deleted M13 ori as shown in SEQ ID NO:18, deletion of 203 nucleotides at position 76-278 compared with wild type M13 ori, length 178 nt, agarose gel electrophoresis as shown in FIG. 3.
EXAMPLE 9 production of circular single stranded DNA by pMT2d5C9MRdM75R16d S
The recombinant phage packaging plasmid pMT2d5C9MRdM R16dS is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 19, the initial sequence with the length of 39 nt,SEQ ID NO:18 is deleted from the 1-290 bit sequence and the 330-381 bit sequence of the full-length initial sequence shown as SEQ ID NO.1, the initial sequence is consistent with 291-329 bit sequences, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 17, the termination sequence with the length of 109 nt,SEQ ID NO:17 is deleted from the 76-278 bit sequence and the 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO. 2, the initial sequence is consistent with 1-75 and 279-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
PMT2d5C9MRdM R16dS-cssDNA total length 4704 nt, wherein the target sequence 4595 nt, the deleted M13 ori is shown in SEQ ID NO:20, the sequence of 203 nucleotides at positions 76-278 and 69 at positions 313-381 are deleted compared with the wild type M13 ori, the length is 109 nt, and agarose gel electrophoresis is shown in FIG. 4.
EXAMPLE 10 production of circular single stranded DNA by pMT2d5C9MRdM83R16
The recombinant phage packaging plasmid pMT2d5C9MRdM R16 was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 21, the termination sequence with the length of 94 nt,SEQ ID NO:21 is deleted from the 68-278 bit sequence and 313-330 bit sequence of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with 1-67 and 279-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1.
The total length of pMT2d5C9MRdM R16-cssDNA is 4765 nt, wherein the target sequence 4595 nt is shown in SEQ ID NO. 22, the deleted M13 ori is deleted by 211 nucleotides at 68-278 positions compared with the wild type M13 ori, the length is 170 nt, agarose gel electrophoresis is shown in FIG. 3, and a sequencing diagram is shown in FIG. 6.
EXAMPLE 11 pMT1d3C2
The recombinant phage packaging plasmid pMT1d3C2 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in sequence. The initial sequence is shown as SEQ ID NO. 1, the length of the initial sequence is 381 nt, the termination sequence is shown as SEQ ID NO. 23, the termination sequence is 262 nt,SEQ ID NO:23, the 263-330 bit sequence of the full-length termination sequence shown as SEQ ID NO.2 is deleted, the sequence is consistent with the 1-262 bit sequence, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1. Agarose gel electrophoresis is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C10 did not successfully package circular single stranded DNA.
EXAMPLE 12 pMT1d5C1
The recombinant phage packaging plasmid pMT1d5C1 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in sequence. The initial sequence is shown as SEQ ID NO.1, the length of the initial sequence is 381 nt, the termination sequence is shown as SEQ ID NO. 24, the termination sequence is 280 nt,SEQ ID NO:24, the 281-330 bit sequence of the full-length termination sequence shown as SEQ ID NO.2 is deleted, the sequence is consistent with the 1-280 bit sequence, and the target sequence is shown as SEQ ID NO. 4. For additional steps reference is made to example 1 and agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT1d5C1 did not successfully package circular single stranded DNA.
EXAMPLE 13 pMT2d5C10
The recombinant phage packaging plasmid pMT2d5C10 was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deletion initial sequence shown as SEQ ID NO. 25, the initial sequence with the length of 81 nt,SEQ ID NO:25 is deleted by 1-300 bits of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with 301-381 bits of sequence, the termination sequence is a deletion termination shown as SEQ ID NO. 6, the termination sequence with the length of 312 nt,SEQ ID NO:6 is deleted by 313-330 bits of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with 1-312 bits of sequence, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1. Agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C10 did not successfully package circular single stranded DNA.
EXAMPLE 14 pMT2d3C1
The recombinant phage packaging plasmid pMT2d3C1 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in sequence. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 26, the initial sequence with the length of 331 nt,SEQ ID NO:26 is deleted from 332-381 bits of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with the sequence from 1-331 bits, the termination sequence is a deleted termination sequence shown as SEQ ID NO. 6, the termination sequence with the length of 312 nt,SEQ ID NO:6 is deleted from 313-330 bits of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with the sequence from 1-312 bits, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1. Agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C10 did not successfully package circular single stranded DNA.
EXAMPLE 15 pMT2d5C9F10
The recombinant phage packaging plasmid pMT2d5C9F10 was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the sequence length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO. 1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination shown as SEQ ID NO. 27, the termination sequence with the sequence length of 302 nt,SEQ ID NO:27 is deleted from the 51-60 bit and 312-330 bit sequences of the full-length termination sequence shown as SEQ ID NO. 2, and is consistent with 1-50 and 61-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. For additional steps reference is made to example 1 and agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C9F10 did not successfully package circular single stranded DNA.
EXAMPLE 16 pMT2d5C9MRdR25
The recombinant phage packaging plasmid pMT2d5C9MRdR was obtained by PCR subcloning and consisted of the starting sequence, the target sequence and the termination sequence in order. The initial sequence is a deleted initial sequence shown as SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 bit sequence of the full-length initial sequence shown as SEQ ID NO.1, and is consistent with 291-381 bit sequence, the termination sequence is a deleted termination shown as SEQ ID NO. 28, the termination sequence with the length of 175 nt,SEQ ID NO:28 is deleted from the 151-287 bit and 312-330 bit sequences of the full-length termination sequence shown as SEQ ID NO.2, and is consistent with 1-150, 288-312 bit sequences, and the target sequence is shown as SEQ ID NO. 4. Other steps refer to example 1. Agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C9MRdR did not successfully package circular single stranded DNA.
EXAMPLE 17 pMT2d5C9MRdM90R16
The recombinant phage packaging plasmid pMT2d5C9MRdM R16 is obtained by PCR subcloning and consists of a starting sequence, a target sequence and a termination sequence in order. The initial sequence is the initial sequence deleted as shown in SEQ ID NO. 5, the initial sequence with the length of 91 nt,SEQ ID NO:5 is deleted from the 1-290 th bit sequence of the full-length initial sequence as shown in SEQ ID NO. 1, and is consistent with 291-381 th bit sequence, the termination sequence is the termination of the deletion as shown in SEQ ID NO. 29, the termination sequence with the length of 94 nt,SEQ ID NO:29 is deleted from the 61-278 th bit and 312-330 th bit sequences of the full-length termination sequence as shown in SEQ ID NO. 2, and is consistent with the 1-60 th bit and 179-312 th bit sequences, and the target sequence is shown in SEQ ID NO. 4. Other steps refer to example 1. Agarose gel electrophoresis of the final product is shown in FIG. 7. As can be seen from FIG. 7, phage packaging plasmid pMT2d5C9MRdM R16 did not successfully package circular single stranded DNA.
EXAMPLE 18 preparation of micro-circular DNA
The css DNA was PCR asymmetrically amplified using a single-primer PCR method with circular single-stranded DNA (pMPdID C-cssDNA) as a template. The double strand of DNA, single strand of DNA, etc. in the product is then digested with exonuclease V (RecBCD), leaving the loop (with nicks) cdsDNA. Finally, the product is purified and quantified by using a column purification mode.
1) CssDNA asymmetric amplification
1.1 Carrying out asymmetric PCR amplification on pMPdID C-cssDNA, wherein an amplification system is shown in Table 3, fully and uniformly mixing after preparation, and eliminating bubbles in the system by short centrifugation with a mini centrifuge. The PCR amplification procedure is described in Table 4.
TABLE 3 cssDNA asymmetric PCR amplification System
TABLE 4 PCR amplification procedure
2) Digestion reaction liquid
The reaction solution obtained in the previous step is digested, the digestion system is shown in Table 5, centrifuged at 5 s in sequence, and put into a 37 ℃ constant temperature incubator for digestion of 30 min.
TABLE 5 digestive system
3) Purifying the reaction solution
The reaction solution is purified by using a common DNA product purification kit of the Tiangen brand, absolute ethyl alcohol is added into a rinsing solution PW before use, and a label on a volume reference bottle is added.
3.1 Column equilibration 500. Mu.L of equilibration solution BL was added to the adsorption column CB2 (the adsorption column was placed in the collection tube), centrifuged at 12,000 rpm for 1 min, and the waste liquid in the collection tube was discarded.
3.2 PB was added to the reaction mixture in an amount of 5 times by volume, followed by thoroughly mixing.
3.3 Adding the solution obtained in the last step into an adsorption column CB2 (the adsorption column is placed in a collecting pipe), standing at room temperature for 2min, at 12,000 rpm, centrifuging for 1 min, pouring out the waste liquid in the collecting pipe, and placing the adsorption column CB2 into the collecting pipe.
3.4 600. Mu.L of the rinse PW was added to the adsorption column CB2, centrifuged at 12,000 rpm for 1 min, and the waste liquid in the collection tube was discarded, and the adsorption column CB2 was placed in the collection tube.
3.5 Repeating the operation step 3.4.
3.6 The adsorption column CB2 was returned to the collection tube and centrifuged at 12,000 rpm for 2 min. Then the adsorption column CB2 is left at room temperature for a few minutes and thoroughly dried.
3.7 An amount of enzyme-free water (about 30-60. Mu.l) was dropped into the adsorption column to elute, left to stand for 2 min, and then centrifuged at 12,000 rpm for 2 min.
3.8 The centrifuged product was detected by agarose gel electrophoresis.
Through the above experimental procedures, the agarose gel electrophoresis result shown in FIG. 8 shows that there is only one bright band after digestion and purification, i.e., the circular single-stranded DNA is changed from single strand to double strand.
The present invention has found that even if there is a certain deletion of the start sequence and the stop sequence of M13ori, it can be successfully used as recombinant phage packaging plasmid for further packaging and finally generating single-stranded DNA. The recombinant phage packaging plasmid provided by the invention has the advantages that the initial sequence can be shortened to 291-329 bits of the sequence shown in SEQ ID NO. 1, and the termination sequence of the recombinant phage packaging plasmid must at least comprise two sections of sequences shown in 1-67 bits and 279-312 bits of SEQ ID NO. 2, so that circular single-stranded DNA can be successfully packaged. Compared with the traditional full-length M13ori sequence, the stationary phase sequence of the circular single-stranded DNA prepared by the recombinant phage packaging plasmid provided by the invention is shorter, the influence of M13ori serving as a framework sequence on a target sequence can be reduced, and compared with the traditional method, the circular single-stranded DNA prepared by the invention is simpler, more convenient and lower in cost.
The above examples illustrate only a few embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that various changes, modifications and substitutions can be made by those skilled in the art without departing from the spirit of the invention, which is intended to be within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Although the present invention has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope of the invention as defined in the claims.
Claims (11)
1. A recombinant phage packaging plasmid, characterized in that the recombinant phage packaging plasmid comprises a starting sequence, a target sequence and a termination sequence, wherein the starting sequence at least comprises an x-v bit sequence in a sequence shown as SEQ ID NO.1 from 5 'to 3', the termination sequence at least comprises a 1-y bit sequence and a z-w bit sequence in a sequence shown as SEQ ID NO. 2, x is not less than 291,329 and not more than 381,67 and y is not less than 279,312 and not more than w is not more than 330, wherein x, y, z, v, w is a positive integer, and the starting sequence is not a sequence shown as SEQ ID NO.1 and/or the termination sequence is not a sequence shown as SEQ ID NO. 2.
2. The recombinant phage packaging plasmid of claim 1, further comprising at least one deoxyribose nucleic acid cleavage site or restriction enzyme cleavage site.
3. The recombinant phage packaging plasmid of claim 1, wherein x=291 and v=329, and wherein the starting sequence comprises at least the 291-329 position of the sequence shown in SEQ ID No. 1.
4. The recombinant phage packaging plasmid of claim 1, wherein said y=67, said z=279, said w=312 and said termination sequence comprises at least the sequences 1-67 and 279-312 of the sequences set forth in SEQ ID No. 2.
5. The recombinant phage packaging plasmid of claim 1, wherein the starting sequence is the sequence shown as SEQ ID NO. 5 or SEQ ID NO. 19.
6. The recombinant phage packaging plasmid of claim 1, wherein the termination sequence is selected from any one of the sequences shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and SEQ ID NO. 21.
7. A method for preparing circular single-stranded DNA, comprising designing and constructing the recombinant phage packaging plasmid of any one of claims 1-6, transforming a host cell with the recombinant phage packaging plasmid and helper plasmid for replication, and extracting circular single-stranded DNA produced in the host cell.
8. A circular single-stranded DNA, characterized in that the circular single-stranded DNA comprises a target sequence and a deleted M13 ori sequence, wherein the deleted M13 ori sequence at least comprises a sequence shown as 1-y and z-329 in SEQ ID NO. 1, wherein 67.ltoreq.y < z.ltoreq.279, and z is an integer at least 2 larger than y.
9. The circular single stranded DNA of claim 8, further comprising at least one deoxyribose or restriction enzyme cleavage site.
10. A method for producing a linear single-stranded DNA, comprising subjecting the circular single-stranded DNA of claim 9 to cleavage with a deoxyribose or a restriction enzyme to obtain a linear single-stranded DNA.
11. A method for preparing a micro-ring DNA, comprising preparing the circular single-stranded DNA according to claim 8 or 9, and performing in vitro complementation on the circular single-stranded DNA to obtain the micro-ring DNA.
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| US20210340571A1 (en) * | 2019-01-04 | 2021-11-04 | Stellate Biotherapeutics, Inc. | Targeted genome modification using circular single-stranded dna |
| CN113950531A (en) * | 2019-05-15 | 2022-01-18 | 麻省理工学院 | Optimization of circular single-stranded DNA using M13 phage |
| CN114269938A (en) * | 2019-06-21 | 2022-04-01 | 阿斯克肋匹奥生物制药公司 | Production of vectors using phage replication origins |
| WO2024206940A1 (en) * | 2023-03-31 | 2024-10-03 | Kano Therapeutics Inc. | Engineered cssdna production hosts and phagemids |
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| US20210340571A1 (en) * | 2019-01-04 | 2021-11-04 | Stellate Biotherapeutics, Inc. | Targeted genome modification using circular single-stranded dna |
| CN113950531A (en) * | 2019-05-15 | 2022-01-18 | 麻省理工学院 | Optimization of circular single-stranded DNA using M13 phage |
| CN114269938A (en) * | 2019-06-21 | 2022-04-01 | 阿斯克肋匹奥生物制药公司 | Production of vectors using phage replication origins |
| WO2024206940A1 (en) * | 2023-03-31 | 2024-10-03 | Kano Therapeutics Inc. | Engineered cssdna production hosts and phagemids |
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