CN105760715A - Image visualization method for detecting single molecule DNA duplication - Google Patents
Image visualization method for detecting single molecule DNA duplication Download PDFInfo
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Abstract
The invention discloses an image visualization method for detecting single molecule DNA duplication. The method comprises the steps that single-chain DNA and a buffering system of a DNA polymerase Klenow fragment combined with the single-chain DNA are scanned with an atomic force microscope, and DNA duplication is subjected to imaging observation through the atomic force microscope, wherein the single-chain DNA contains dNTP, and the two ends of the single-chain DNA are fixed to two different positions on hollow DNA folded paper respectively. According to the method, the DNA duplication process is tracked on the single molecule level, the whole process of combination of the single-chain DNA and single DNA polymerase, moving between the DNA chain and the DNA polymerase and conversion from the single-chain DNA chain to a double-chain DNA chain is recorded, various forms of state distribution of the DNA chain on the surface of a substrate and the changing situation in the interaction process of DNA and the DNA polymerase can be captured, the biomolecule behavior action mechanism can be understood more easily, and data on the time scale and the space scale in the molecule event obtaining process can be obtained more easily.
Description
Technical field
The present invention relates to field of nanometer technology, detect, more particularly to a kind of, the image viewing method that unique DNA replicates.
Background technology
DNA replication dna is primarily referred to as with a DNA single chain for antigene strand, according to AT and CG pair principle, synthesizes the process of an other DNA single chain.In vivo, DNA replication dna is extremely important, and the generation of organism, and the life process such as development and evolution is closely related.In vitro, also relate to numerous modern biotechnology and include DNA sequencing, forensic medical examination, nano-biosensing detection and widely used DNA cloning technology etc..
DNA replication dna is a kind of biomolecular reaction process, adopts advanced FRET (fluorescence resonance energy transfer) (FRET) technology and optical tweezer technology, has been obtained for the dynamic process information such as DNA replication dna speed etc. of many DNA replication dnas reaction.The research of image viewing can obtain the data on the time scale of molecular events process and space scale, can provide reliable information for the process of DNA replication dna and Mechanism Study.But in image viewing, on single molecules level, only have the relevant report of the image viewing method for rna transcription at present, and for the DNA replication dna of single molecules level owing to lacking direct observational technique, with pictorial form the dynamic process of DNA replication dna is described and characterizes and cannot solve always.
Summary of the invention
The technical problem to be solved is to lack a kind of method directly with graphic form, the dynamic process of DNA replication dna being described and being characterized in prior art to overcome, provide a kind of image viewing method detecting unique DNA duplication, the nanotechnology DNA paper folding that the atomic force microscopy (AFM) with nanoscale imaging level gets up with new development is combined by the method for the present invention, individual molecule level is followed the tracks of the reproduction process of DNA, record wall scroll DNA be combined with each other with single DNA polymerase, movement between DNA and archaeal dna polymerase, and finally single stranded DNA chain is transformed into the overall process of double-stranded DNA chain.Simultaneously, also can catch the various distributions of DNA on substrate surface, and situation of change when DNA and archaeal dna polymerase interaction, contribute to understanding biomolecule behavior mechanism of action, and the data on the time scale of molecular events process and space scale can be obtained, process and Mechanism Study for DNA replication dna provide reliable information.
The present invention solves above-mentioned technical problem by the following technical programs:
One of technical solution of the present invention: a kind of image method for visualizing detecting unique DNA duplication, said method comprising the steps of: to containing dNTP, two ends are individually fixed in the single stranded DNA of two diverse locations in hollow DNA paper folding and the buffer system in conjunction with the archaeal dna polymerase Klenow fragment of described single stranded DNA carries out afm scan, by the duplication of described atomic force microscope imaging DNA.
In the present invention, described hollow DNA paper folding can be the hollow DNA paper folding that this area is conventional, including various difform DNA paper foldings, such as rectangle or the hollow DNA paper folding of triangle, it is preferably the equilateral triangle of central hollow, more preferably for the equilateral triangle of central hollow that the interior length of side is 30-50nm, best for the equilateral triangle of central hollow that the interior length of side is 40-50nm, such as the equilateral triangle of central hollow that the interior length of side is 50nm.The pattern of described hollow DNA paper folding can design as required, and the centre of a desirable pattern exists space can hold distance and the archaeal dna polymerase for the treatment of to detect DNA height simultaneously.The preparation method of described hollow DNA paper folding can be the method that this area is conventional, preferably it is self-assembly of by strand scaffold chain DNA and staple single stranded DNA, staple single stranded DNA is used for fixing scaffold chain DNA and forms targeted graphical, more preferably comprises the following steps: the staple chain DNA set ABCL that strand two chains of scaffold chain DNA M13mp18 and A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is mixed in TAE-Mg2+Buffer system, it is annealed to 20 DEG C from 95 DEG C, annealing rate is 0.1 DEG C/10s, wherein, described strand scaffold chain M13mp18 and two chains of described A17 and C33 replace with the ratio that the molar concentration rate of the staple chain DNA set ABCL of sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 can be this area routine respectively, it is preferred that be 1: 10.The DNA paper folding 40mMTris-acetic acid that assembles, 1mMEDTA, 12.5mMMgCl2, pH8.0 buffer ultrafiltration.nullDescribed staple chain DNA set ABCL is described staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page，Always have 208 sequences，Equilateral triangle DNA paper folding can be self-assembled into，Author is DingB，DengZ，YanH，CabriniS，ZuckermannRN，BokorJ，Described JAmChemSoc. is " American Chemical Society's periodical ".
In the present invention, described single stranded DNA (ssDNA) can be the single stranded DNA that this area is conventional, and it is combined by described archaeal dna polymerase Klenow fragment as the template of DNA replication dna.Described single stranded DNA can be fixed on two diverse locations of described hollow DNA paper folding by the mode that this area is conventional, preferably, described single stranded DNA is fixed in described hollow DNA paper folding by the sequence hybridization pairing respectively have more a section complementary with 3 ' ends with described single stranded DNA 5 ' end respectively with on two diverse locations in described hollow DNA paper folding inside edge and is positioned at the central authorities of described hollow DNA paper folding, or 5 ' ends of described single stranded DNA and 3 ' ends be separately fixed at described hollow DNA paper folding outside same on two diverse locations.When described single stranded DNA is fixed in described hollow DNA paper folding and is positioned at described hollow DNA paper folding central, the different position of said two is not inside described hollow DNA paper folding on collinear edge, but can lay respectively on the edge of different straight line, such as lay respectively on two limits in Atria bar limit, or be arranged on two limits of rectangle four edges；More preferably, 20 bases that described single-stranded DNA templates is held with the 5 ' of staple chain SEQIDNO.17 respectively by its 5 ' end and its 3 ' end are fixed in described DNA paper folding with the 3 ' of staple chain SEQIDNO.98 20 the base hybridization held and are positioned at described DNA paper folding central authorities.Described hybridization can be the method that this area is conventional, it is preferred that for being annealed to 15 DEG C with the speed of 0.5 DEG C/min after being warming up to 50 DEG C after being mixed with described hollow DNA paper folding by described single stranded DNA, then uses TAE-Mg2+Buffer ultrafiltration.The final concentration of described dNTP can be the concentration making DNA synthesis to be normally carried out that this area is conventional, it is preferred that be 15.6 μMs.Described buffer system can be the conventional buffer system of this area archaeal dna polymerase Klenow fragment work, and the buffer composition in described buffer system is preferably TAE-Mg2+Buffer, described TAE-Mg2+Buffer is 40mMTris-acetic acid, 1mMEDTA, 12.5mMMgCl2, pH8.0.When the 5 ' ends and 3 ' of described single stranded DNA hold two diverse locations on the same one side in the outside being separately fixed at described hollow DNA paper folding, the single stranded DNA formed after the staple chain of the strand scaffold DNA disappearance pairing in the outside on the limit that described single stranded DNA is preferably in described hollow DNA paper folding.
In the present invention, described buffer system is carried out the operation that afm scan can be this area routine, preferably sequentially include the following steps: 1) described single stranded DNA two ends are individually fixed in two diverse locations in described hollow DNA paper folding, prepare single stranded DNA-hollow DNA paper folding；2) by step 1) described single stranded DNA-hollow DNA paper folding drop to new explanation from Muscovitum on, after adsorbing 3-5 minute, AFM imaging；3) after obtaining single stranded DNA-hollow DNA paper folding image, the mixed liquor of archaeal dna polymerase Klenow fragment and dNTPs is added, simultaneously scanning imagery under an atomic force microscope.
nullStep 1) in，Described single stranded DNA two ends are individually fixed in described hollow DNA paper folding two diverse locations preferably can be undertaken by the method comprised the following steps: is fixed in described hollow DNA paper folding with 3 ' end hybridization by 5 ' ends by described single stranded DNA and is arranged in the hollow of described DNA paper folding central authorities，Or when described hollow DNA paper folding assembles, the staple chain part in outside on one side is cancelled or shortened，The strand scaffold DNA of the staple chain pairing made and be shortened or cancel is not paired and keeps original single stranded DNA state，Form single stranded DNA，More preferably undertaken by the method comprised the following steps: select two diverse locations respectively to have more one section of nucleotide sequence in the staple chain-ordering of the inside edge of described hollow DNA paper folding and match with 3 ' terminal sequences with 5 ' ends of described single-stranded DNA templates respectively，Described single-stranded DNA templates is made to be fixed in described DNA paper folding by 5 ' ends and 3 ' end hybridization and be positioned at described DNA paper folding central authorities.
Step 2) in, the time of described absorption is 3-5 minute, it is preferred that be 3 minutes.
Step 3) in, the final concentration of described dNTP can be the concentration making DNA synthesis to be normally carried out that this area is conventional, it is preferred that be 15.6 μMs.
In the present invention, described atomic force microscope can be the atomic force microscope that this area is conventional, described atomic force microscope probe can be the atomic force microscope probe that this area is conventional, it is preferred that for Bruke company silicon nitrate probes (SNL-10, coefficient of elasticity 0.35N/m).The scan pattern of described atomic force microscope can be the pattern that this area is conventional, for example with " J " probe, " rap " pattern, regulate sweep parameter in little Li Qu (F < 200pN) imaging, collect the afm image of different phase sample respectively.The substrate of described atomic force microscope imaging be new explanation from Muscovitum.During use, the described single stranded DNA being fixed in described DNA paper folding is placed on described mica surface, and described Muscovitum sticks on iron plate, is namely placed on atomic force microscope load sample platform afterwards and carries out atomic force microscope imaging.The time of described scanning can be the duration that this area is conventional, it is preferred that for 1-8min, is more preferably 4.5-8min, is more preferably 6.5-8min further, is 8min best.
In the present invention, the described duplication by described atomic force microscope imaging DNA can be undertaken by the method that this area is conventional, it is preferably and is undertaken by analysis AFM feature image and height map, the such as reference value using the height of described DNA paper folding as the double-stranded DNA height of synthesis, measure to the height of DNA being positioned at described DNA paper folding central authorities be described DNA paper folding highly more than 1/2 be considered as double-stranded DNA, less than 1/2 be considered as single stranded DNA.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can combination in any, obtain the preferred embodiments of the invention.
Present invention also offers a preferred embodiment, it comprises the following steps:
(1) strand two chains of scaffold chain DNA M13mp18 and A17 and C33 are replaced with the set ABCL of sequence staple chain DNA shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively and be mixed in TAE-Mg2+Buffer system, is annealed to 20 DEG C from 95 DEG C, and annealing rate is 0.1 DEG C/10s, prepares hollow DNA paper folding；nullWherein said staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page，The molar concentration rate of the staple chain DNA set ABCL that described strand scaffold chain M13mp18 and two chains of described A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is 1: 10，Described TAE-Mg2+Buffer is 40mMTris-acetic acid, 1mMEDTA, 12.5mMMgCl2, pH8.0；
(2) the hollow DNA paper folding TAE-Mg that step (1) is prepared2+Buffer system ultrafiltration, obtains the hollow DNA paper folding of ultra filtration；
(3) the hollow DNA paper folding of the sequence such as single stranded DNA shown in sequence table SEQ IDNO.3 and ultra filtration described in step (2) is mixed in molar ratio at 50: 1, it is warming up to 50 DEG C, it is annealed to 15 DEG C, with aforementioned TAE-Mg with the annealing rate of 0.5 DEG C/min2+Buffer ultrafiltration, obtains single stranded DNA-hollow DNA paper folding；
(4) by step 3) described single stranded DNA-hollow DNA paper folding drop to new explanation from Muscovitum on, after adsorbing 3-5 minute, atomic force microscope imaging；Described atomic force microscope probe is silicon nitrate probes SNL-10, and its coefficient of elasticity is 0.35N/m；The scan pattern of described atomic force microscope adopts " J " probe, " rapping " pattern, regulates the sweep parameter little Li district imaging at F < 200pN；The time of described scanning is 0-8 minute.
Agents useful for same of the present invention and raw material are all commercially.
The actively progressive effect of the present invention is in that: the nanotechnology DNA paper folding that the atomic force microscopy (AFM) with nanoscale imaging level gets up with new development is combined by detection method provided by the invention, individual molecule level is followed the tracks of the reproduction process of DNA, movement between record wall scroll DNA and the be combineding with each other of single DNA polymerase, DNA and archaeal dna polymerase, and final single stranded DNA chain is transformed into the overall process of double-stranded DNA chain.Simultaneously, also can catch the various distributions of DNA on substrate surface, and situation of change when DNA and archaeal dna polymerase interaction, contribute to understanding biomolecule behavior mechanism of action, and the data on the time scale of molecular events process and space scale can be obtained, process and Mechanism Study for DNA replication dna provide reliable information.
Accompanying drawing explanation
Fig. 1 is the single stranded DNA-DNA paper folding schematic diagram of embodiment 1 and embodiment 2.
Fig. 2 is the schematic diagram that the present invention passes through afm scan single stranded DNA-DNA paper folding detection DNA replication dna
Fig. 3 is the atomic force microscope images of embodiment 3 single DNA molecules reproduction process.Enlarged drawing picture in Fig. 3 A1, A2, B1, B2, E1 and E2 respectively Fig. 3 A, B and E frame and height image；Enlarged drawing picture in Fig. 3 C1, C2, D1 and D2 respectively Fig. 3 C and D center and height image.
Fig. 4 is the embodiment 4DNA FRET (fluorescence resonance energy transfer) collection of illustrative plates replicated and kinetic curve figure.
Fig. 5 is the atomic force microscope images that embodiment 5 single DNA molecules replicates.
Detailed description of the invention
Mode by the examples below further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Reagent and instrument source in the present invention are as follows:
M13mp18 single stranded DNA and archaeal dna polymerase fragment Klenow fragment (KF) are purchased from NEB company；
Staple chain is purchased from Sangon Biotech (Shanghai) Co., Ltd.；
DNTPs is purchased from TaKaRa company (China, Dalian)；
Cy5-dCTP is purchased from GE (U.S.)
Atomic force microscope probe is Bruke company silicon nitrate probes (SNL-10, coefficient of elasticity 0.35N/m)；
PCR instrument is PTC-200PeltierThermalCycler (MJResearch)；
AFM is NANOScopeIIIa or NANOScopeVIII system (DigitalInstrument, the U.S.).
In the present invention, used staple chain DNA set ABCL is described staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page, always have 208 DNA sequence, equilateral triangle DNA paper folding can be self-assembled into.
The preparation of embodiment 1 single stranded DNA-DNA paper folding
The hollow DNA paper folding of the present embodiment design utilizes M13mp18DNA and staple strand to be self-assembly of equilateral triangle pattern, and the preparation method of equilateral triangle hollow DNA paper folding is as follows: the staple chain DNA set ABCL that annular long two chains of scaffold single stranded DNA M13mp18 and A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is mixed in TAE-Mg2+Buffer system (40mMTris-acetic acid, 1mMEDTA, 12.5mMMgCl2PH8.0), it is placed in PCR instrument again and is annealed to 20 DEG C from 95 DEG C, annealing rate is 0.1 DEG C/10s, wherein the molar concentration rate of the staple chain DNA set ABCL that two chains of M13mp18 and A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is 1: 10, uses TAE-Mg2+Buffer ultrafiltration, prepares the hollow DNA paper folding of equilateral triangle assembled, and the length of side is about 120nm.The staple chain A2 on A limit of the hollow DNA paper folding of equilateral triangle of gained, A3, A6, A7, A10 and A11 intermediate sequence respectively have more 28 bases (5 '-the TCCTCTTTTGAGGAACAAGTTTTCTTGT-3 ') marker as DNA paper folding, 5 ' the ends of staple chain A17 stretch out 20 bases (CGTAGGAGTCCTACACTACC) more, and the 3 ' of C33 are held and stretched out 20 bases (TAACACTCCGAACGTATTAC) for the template DNA hybridization at paper folding ad-hoc location more.
The sequence such as 114ntDNA template shown in sequence table SEQ IDNO.3 is hybridized in above-mentioned prepared hollow DNA paper folding, so as to be positioned at hollow DNA paper folding central authorities, its two ends are fixed in hollow DNA paper folding by hybridization, form single stranded DNA-hollow DNA paper folding, step is as follows: 114ntDNA template and hollow DNA paper folding is mixed is placed on PCR instrument at 50: 1 in molar ratio, being annealed to 15 DEG C from 50 DEG C, annealing rate is 0.5 DEG C/min, uses TAE-Mg2+Preparing single stranded DNA-hollow DNA paper folding after buffer ultrafiltration, structural representation is as shown in Figure 1A.After measured, the molar concentration of obtained single stranded DNA-hollow DNA paper folding is about 2nM.
The preparation of embodiment 2 single stranded DNA-DNA paper folding
nullThe hollow DNA paper folding of the present embodiment design utilizes M13mp18DNA and staple chain DNA set ABCL to be self-assembly of equilateral triangle pattern，Wherein，The staple chain part on the C limit of described equilateral triangle shortens，Specifically，By C08、C12、C16、C20、C23、C24、C26、C28、C30、C41、C45、C49、C53、C56、C59、C61、C63 and C64 replaces with sequence such as sequence table SEQ IDNO.4 respectively、SEQIDNO.5、SEQIDNO.6、SEQIDNO.7、SEQIDNO.8、SEQIDNO.9、SEQIDNO.10、SEQIDNO.11、SEQIDNO.12、SEQIDNO.13、SEQIDNO.14、SEQIDNO.15、SEQIDNO.16、SEQIDNO.17、SEQIDNO.18、SEQIDNO.19、Sequence shown in SEQIDNO.20 and SEQIDNO.21，The M13mp18 single stranded DNA making appropriate section does not match，Form outer side edges and become single stranded DNA along a DNA，The component of other assembly systems and method are with embodiment 1，The structural representation of obtained single stranded DNA-DNA paper folding is as shown in Figure 1B.After measured, the molar concentration of obtained single stranded DNA-hollow DNA paper folding is about 2nM.
DNA replication dna is carried out the image viewing detection analysis of Real-time and Dynamic by embodiment 3 by atomic force microscope
Triangle single stranded DNA obtained by embodiment 1-hollow DNA paper folding 3.0 μ L dropping is placed 3 minutes from mica surface in new explanation, after liquid cell adds 30 μ LTAE buffer, use afm scan imaging, obtaining the nanostructured of DNA paper folding clearly and the image of DNA profiling chain, ideograph is as shown in Figure 2.Subsequently, the mixed solution containing the archaeal dna polymerase Klenow fragment that 2 μ L concentration are 85U/mL and dNTPs that 2 μ L concentration are 62.5 μMs is joined in liquid cell, scanning imagery immediately.Scanning adopts " J " probe, " rapping " pattern, regulates sweep parameter in little Li Qu (F < 200pN) imaging.After adding the mixed liquor containing Klenow fragment and dNTPs, 1min, 4.5min, 6.5min and 8min collect the atomic force microscope images of sample respectively, and image NanoScopeAnalysis software carries out processing and analyzing.
Result is as it is shown on figure 3, Fig. 3 A-C shows that Klenow fragment combines with template strand, and Fig. 3 C-D demonstrates Klenow fragment and moves on template strand, and Fig. 3 E shows that Klenow fragment is dissociated from DNA.
Embodiment 4 FRET (fluorescence resonance energy transfer) (FRET) verifies that result is analyzed in the image viewing detection of embodiment 3
Preparing single stranded DNA-DNA paper folding by the method for embodiment 1, be wherein marked with Cy3 fluorescence group at the 5 ' end positions of staple chain SEQIDNO.17, other reagent and method are identical with embodiment 1.
Prepared single stranded DNA-DNA paper folding is mixed with the Klenow fragment of inactivation with active Klenow fragment respectively with Cy5-dCTP, dTTP, dATP, dGTP, make each component final concentration respectively 0.5mMdNTPs, 0.05U/ μ LKF and 0.1nM single stranded DNA-DNA paper folding, two groups of systems are put in fluorescence spectrophotometer detect at once.Spectra collection Data Source is in HitachiF-4500 spectrofluorophotometer.After adding reactive component, record each fluorescence data at once, including dynamic process.Spectral scan parameter Cy3 is Ex550nm and Em570；Cy5 is Ex649nm and Em670nm, sweeps speed for 30000nm/min.
Result as shown in Figure 4 A, when FRET system exists the Klenow fragment of Cy3, Cy5, dNTP and inactivation, Cy3 fluorescence signal is strong and Cy5 fluorescence signal is weak.When there is Cy3, Cy5, dNTP and active Klenow fragment in FRET system, Cy5 fluorescence signal increases, and reduces with Cy3 fluorescence signal simultaneously.The change of this fluorescence intensity level, describes the generation of FRET (fluorescence resonance energy transfer) event, demonstrates in embodiment 3 single stranded DNA in reaction system and copies as double-stranded DNA.Simultaneously as shown in Figure 4 B, Cy5 fluorescence intensity changes over curve chart and shows that Cy5 fluorescence intensity changes over and be stepped up, it was demonstrated that the generation of DNA replication dna.
DNA replication dna is carried out the image viewing detection analysis of Real-time and Dynamic by embodiment 5 by atomic force microscope
Triangle single stranded DNA obtained by embodiment 2-hollow DNA paper folding 3.0 μ L dropping is placed 5 minutes from mica surface in new explanation, after liquid cell adds 40 μ LTAE buffer, use afm scan imaging, it is thus achieved that the image of the nanostructured of DNA paper folding clearly and DNA profiling chain.Subsequently, by containing joining in liquid cell after to be the archaeal dna polymerase Klenow fragment of 170U/mL, 5 μ L concentration be dNTPs and the 30 μ LKlenow fragment buffer mixing of 2500 μMs of 0.2 μ L concentration, scanning imagery immediately.Scanning adopts " J " probe, " rapping " pattern, regulates sweep parameter in little Li Qu (F < 200pN) imaging.Scanning 2 hours continuously after adding the mixed liquor containing Klenow fragment and dNTPs, collect the atomic force microscope images of part-time point sample respectively, image NanoScopeAnalysis software carries out processing and analyzing.
Result is as it is shown in figure 5, reflect the afm image of archaeal dna polymerase Klenow fragment examinations in DNA paper folding, and figure a-f is from DNA replication dna initial reaction stage to the afm image in reaction later stage successively.The height of line place in figure a and f has been carried out detection and has analyzed by figure g and h respectively, the height that wherein figure g display figure a hollow DNA paper folding edge single stranded DNA line place is corresponding is 0.9nm, and the height scheming h display figure f hollow DNA paper folding edge single stranded DNA line place corresponding is 2.3nm, more than in figure a 2 times of single stranded DNA height, show that the duplication of the single stranded DNA of line place in figure f has occurred and that, define double-stranded DNA.
Should be understood that, after the foregoing having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (11)
1. one kind is detected the image method for visualizing that unique DNA replicates, it is characterized in that, said method comprising the steps of: to containing dNTP, two ends are individually fixed in the single stranded DNA of two diverse locations in hollow DNA paper folding and the buffer system in conjunction with the archaeal dna polymerase Klenow fragment of described single stranded DNA carries out afm scan, by the duplication of described atomic force microscope imaging DNA.
2. method as claimed in claim 1, it is characterised in that described hollow DNA paper folding is equilateral triangle.
3. method as claimed in claim 1, it is characterised in that described hollow DNA paper folding is self-assembly of by strand scaffold chain DNA and staple single stranded DNA, and staple strand is used for fixing scaffold chain formation targeted graphical.
null4. method as claimed in claim 3，It is characterized in that，Described strand scaffold chain DNA is M13mp18，The nucleotides sequence of described staple single stranded DNA is classified as two chains of A17 and C33 and replaces with the staple chain DNA set ABCL of sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively，Described staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page；The nucleotide sequence of described single stranded DNA is such as shown in sequence table SEQ IDNO.3.
5. method as claimed in claim 4, it is characterized in that, 20 bases that described single stranded DNA is held with the 5 ' of staple chain SEQIDNO.1 respectively by its 5 ' end and its 3 ' end are fixed in described DNA paper folding with the 3 ' of staple chain SEQIDNO.2 20 the base hybridization held and are positioned at described DNA paper folding central authorities.
6. method as claimed in claim 5, it is characterised in that described hybridization is be annealed to 15 DEG C with the speed of 0.5 DEG C/min after being warming up to 50 DEG C after being mixed with described hollow DNA paper folding by described single stranded DNA, then uses TAE-Mg2+Buffer ultrafiltration.
7. as claimed in claim 1 method, it is characterised in that 5 ' ends of described single stranded DNA and 3 ' ends be separately fixed at the outside of described hollow DNA paper folding same on two diverse locations.
8. method as claimed in claim 7, it is characterised in that described single stranded DNA is the single stranded DNA formed after the staple chain of the strand scaffold DNA disappearance pairing in the outside on a limit in described hollow DNA paper folding.
9. method as claimed in claim 1, it is characterised in that described buffer system is carried out afm scan and comprises the following steps:
1) described single stranded DNA two ends are individually fixed in two diverse locations in described hollow DNA paper folding, prepare single stranded DNA-hollow DNA paper folding；Preferably, the staple chain-ordering of the inside edge of described hollow DNA paper folding selects two diverse locations respectively have more one section of nucleotide sequence to match with 3 ' terminal sequences with 5 ' ends of described single-stranded DNA templates respectively, make described single-stranded DNA templates hold by 5 ' and be fixed in described DNA paper folding with 3 ' end hybridization and be positioned at described DNA paper folding central authorities；Described hollow DNA paper folding is preferably prepared by the method comprised the following steps: the staple chain DNA set ABCL that strand two chains of scaffold chain DNA M13mp18 and A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is mixed in TAE-Mg2+nullBuffer system，It is annealed to 20 DEG C from 95 DEG C，Annealing rate is 0.1 DEG C/10s，Wherein said staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page，The molar concentration rate of the staple chain DNA set ABCL that described strand scaffold chain M13mp18 and two chains of described A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is 1: 10，Described TAE-Mg2+Buffer is 40mMTris-acetic acid, 1mMEDTA, 12.5mMMgCl2, pH8.0, the nucleotide sequence of described single stranded DNA is preferably as shown in sequence table SEQ IDNO.3；
2) by step 1) described single stranded DNA-hollow DNA paper folding drop to new explanation from Muscovitum on, after adsorbing 3-5 minute, atomic force microscope imaging；
3) step 2 is being obtained) after described single stranded DNA-hollow DNA paper folding image, add the mixed liquor of archaeal dna polymerase Klenow fragment and dNTPs, scanning imagery under an atomic force microscope simultaneously.
10. method as claimed in claim 1, it is characterised in that the scanning mould of described atomic force microscope adopts " J " probe, " rapping " pattern, regulates sweep parameter in F < 200pN little Li district's imaging；And/or, the substrate of described atomic force microscope imaging is Muscovitum；And/or, the time of described scanning is 1-8 minute.
11. method as claimed in claim 1, it is characterised in that said method comprising the steps of:
(1) the staple chain DNA set ABCL that strand two chains of scaffold chain DNA M13mp18 and A17 and C33 replace with sequence respectively shown in sequence table SEQ IDNO.1 and SEQIDNO.2 is mixed in TAE-Mg2+Buffer system, is annealed to 20 DEG C from 95 DEG C, and annealing rate is 0.1 DEG C/10s, prepares hollow DNA paper folding；nullWherein said staple chain DNA set ABCL is the set of the DNA sequence from A01 to Loop described in S11-S16 page that on March 17th, 2010 is published in the SupportinOnlineInformation of the paper being entitled as Goldnanoparticleself-similarchainstructureorganizedbyDNA origami of JAmChemSoc. the 132nd volume the 10th phase 3248-3249 page，The molar concentration rate of the staple chain DNA set ABCL that described strand scaffold chain M13mp18 and two chains of described A17 and C33 replace with sequence shown in sequence table SEQ IDNO.1 and SEQIDNO.2 respectively is 1: 10，Described TAE-Mg2+Buffer is 40mMTris-acetic acid, 1mMEDTA, 12.5mMMgCl2, pH8.0；
(2) the hollow DNA paper folding TAE-Mg that step (1) is prepared2+Buffer system ultrafiltration, obtains the hollow DNA paper folding of ultra filtration；
(3) the hollow DNA paper folding of the sequence such as single stranded DNA shown in sequence table SEQ IDNO.3 and ultra filtration described in step (2) is mixed in molar ratio at 50: 1, it is warming up to 50 DEG C, it is annealed to 15 DEG C, with aforementioned TAE-Mg with the annealing rate of 0.5 DEG C/min2+Buffer ultrafiltration, obtains single stranded DNA-hollow DNA paper folding；
(4) by step 3) described single stranded DNA-hollow DNA paper folding drop to new explanation from Muscovitum on, after adsorbing 3-5 minute, atomic force microscope imaging；Described atomic force microscope probe is silicon nitrate probes SNL-10, and its coefficient of elasticity is 0.35N/m；The scan pattern of described atomic force microscope adopts " J " probe, " rapping " pattern, regulates the sweep parameter little Li district imaging at F < 200pN；The time of described scanning is 0-8 minute.
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