CN104388563A - Method for using DNA tetrahedron as scaffold on nano-particle surface and initiating rolling circle amplification reaction - Google Patents
Method for using DNA tetrahedron as scaffold on nano-particle surface and initiating rolling circle amplification reaction Download PDFInfo
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Abstract
The invention relates to a method for using a DNA tetrahedron as a scaffold on a nano-particle surface and initiating rolling circle amplification reaction; after the prepared DNA tetrahedron is assembled on the nano-particle surface, a section of single-stranded DNA extending on the DNA tetrahedron can be used as primer DNA; circular DNA complementary with a special aptamer sequence is used as an amplification template; the circular DNA is hybridized with the primer DNA while the rolling circle amplification reaction is initiated; and the prepared product is multiple long single-stranded DNA composed of multiple apatmer structures on the nano-particle surface. The method disclosed by the invention is capable of effectively regulating and controlling the DNA distribution density on the nano-particle surface, so that the hybridization efficiency of the primer DNA and the circular DNA is increased; by means of long single-stranded DNA composed of the multiple aptamer structures prepared through the rolling circle amplification technology, the CTC (Carbon Tetra Choride) capture efficiency is greatly increased; and thus, the method disclosed by the invention has potential application value in the research fields, such as cell detection, cell capture, cell imaging and the like.
Description
Technical field
The invention belongs to nano material functionalization and DNA nanotechnology Application Areas, the DNA tetrahedron relating to a kind of DNA self-assembly as bracket assembled at nano-material surface, and one section of single stranded DNA that DNA tetrahedron extends causes rolling circle amplification reaction, products therefrom is the long single stranded DNA of nanoparticle surface load many specific identification performances, and this structure can be used for cell detection, catch and the research such as cell imaging.
Background technology
Malignant tumour has become one of major disease that mortality ratio is the highest in the world at present, and along with the rising increasingly of tumor incidence, people for the understanding of malignant tumour and the research of research also in continuous intensification: early stage, efficient and specific CTC catches a powerful measure of the early diagnosis being not only tumour, obtain CTC by release and further investigate for its biological characteristics simultaneously, being also conducive to the formulation that risk assessment in the past and resolution occur metastases.And at present there are some problems for the capture technique of CTC, as capture technique efficiency is high thus cause that CTC is more difficult to be detected because of CTC rare numbers reason; In addition, in acquisition procedure, certain damage is produced to CTC activity, affect the biological characteristic research etc. in its later stage.Therefore, how to realize CTC efficient capture and effectively release obtain biological characteristics completely CTC be life science problem urgently to be resolved hurrily in current metastases research.
Tumour-specific Aptamer(aptamer) research rise gradually in recent years and enrich constantly, it obtains one section of single stranded DNA or RNA with part Exponential enrichment phyletic evolution (SELEX) technology screening, the space structure formed by means of himself and target molecules specific recognition, possess that target molecule is wide, avidity is high, the various advantages energy not available for traditional biological antibody such as high specificity, easily transformation modifications, for cancer target research with treat and provide the desirable targets identification molecule of a class.But simultaneously, although the aptamer being assembled in material surface can tumor cell surface receptor specifically, the assembling area limited due to magnetic nano particle sub-surface and aptamer all make the identification of CTC and capture rate have much room for improvement at the lodging assembled state that particle surface is possible.Rolling circle amplification can make to be assembled in nano-material surface DNA sequence and realize copying, and obtains long single stranded DNA product, and single stranded DNA product is made up of a repeated fragment.But the DNA density domination of nano-material surface is improper, not only cause amplification efficiency not high, and the long single stranded DNA product that obtains is wound around mutually, and causes gathering to make Receptor recognition become difficulty.
And the amplification problem that DNA tetrahedron is DNA provides possible solution path.It is matched by six DNA strands to form, and has high structural stability and rigidity.Research shows, DNA tetrahedron effectively can improve the homogeneity that DNA probe arranges in surface arrangement, and the distance between accuracy controlling probe, thus significantly improve sensitivity and the specificity of biological detection.Therefore can on the basis making full use of the distinctive advantage with Development of Nanotechnology, be expected to provide new efficient detection means for the early diagnosis of tumour and provide strong theoretical basis and technical basis for life science problems such as the transfer generation of studying tumour and development.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides and a kind ofly cause the method for rolling circle amplification at nano-material surface DNA tetrahedron as reaction support.
Two of object of the present invention is by rolling circle amplification, and amplification obtains the long single stranded DNA of many aptamer structure, for the efficient capture of CTC.
A kind of nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction, it is characterized in that, after the DNA tetrahedron prepared is assembled in nanoparticle surface, one section of single stranded DNA that DNA tetrahedron extends can be used as primed DNA, use with the annular DNA of specific aptamer complementary as amplification template, hybridize with primed DNA and cause rolling circle amplification and react, the product of preparation is many long single stranded DNAs of nanoparticle surface many apatmer structure composition, be used as cell detection by the combination of aptamer and cell surface special receptor, catch and cell imaging probe.
Described DNA tetrahedron be four single stranded DNAs by DNA hybridization, a kind of tetrahedral structure of self-assembly.
Described nanoparticle is the one in nanometer gold, quantum dot, magnetic nano-particle.
Described DNA tetrahedron be assembled in nanoparticle surface be the DNA tetrahedron of sulfydryl modification by Au-S key, or NH
2the DNA tetrahedron of-modification is assembled in nanometer gold surface by condensation reaction etc. or has the nanoparticle surface containing-COOH base.
One section of single stranded DNA of described extension is the section of DNA sequence of 10-30 base.
Described specific aptamer sequence be acceptor deposit in case can with the section of DNA sequence of its specific binding, as the aptamer structure that can combine with circulating tumor cell surface receptor (EpCAM).
Described amplification template is the DNA unrelated sequences containing one section of non-aptamer complementation.
Many long single stranded DNAs of described many apatmer structure composition are the DNA single chain that aptamer structure nothing to do with train interval occurs, unrelated sequences can reduce the steric effect of aptamer and receptors bind.
The present invention can Effective Regulation nanoparticle surface DNA distribution density thus improve the hybridization efficiency of primed DNA and circular DNA, the efficient bioprobe of constructing function for cell detection, catch and cell imaging field.
Embodiment
Embodiment 1:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.The DNA tetrahedron PAGE electrophoresis prepared and AFM carry out reclaiming, verifying.
Embodiment 2:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-HS-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-HS-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-HS-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.The DNA tetrahedron PAGE electrophoresis prepared and AFM carry out reclaiming, characterizing.
20 μ L (10 μMs) DNA tetrahedral structure is added in 1mL 15nm gold size solution, 100mM PB buffer (pH7.4) is added after shaken at room temperature spends the night, final concentration is 10mM, Ageing solution (10 mM PB are added after ambient temperature overnight, 2M NaCl, pH 7.4) final concentration of salt is 0.15M in solution.After mixture solution ambient temperature overnight, 4 ° of C 12000rpm/min, 15min, after centrifuge washing 3 times, throw out is in 200 μ L, pH7.4 PB(10 mM, 0.15 M NaCl) resuspension in solution, 4 DEG C of preservations.The DNA tetrahedron AFM prepared carries out reclaiming, characterizing.
Embodiment 3:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-NH
2-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-NH
2-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-NH
2-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.DNA tetrahedron PAGE and AFM prepared carries out reclaiming, characterizing.20 μ L (10 μMs) DNA tetrahedral structure in 10 μ L magnetic beads solution (1mM), 37 DEG C of shaken overnight are placed on magnet stand, magnetic levitation, reject supernatant (the DNA tetrahedron do not connected), add 100 μ LpH7.4 PB(10 mM, 0.15 M NaCl) wash three times.Resuspension in 20 μ L PB buffer solution is added, 4 DEG C of preservations in throw out.
Embodiment 4:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-HS-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-HS-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-HS-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.DNA tetrahedron PAGE and AFM prepared carries out reclaiming, characterizing.
20 μ L (10 μMs) DNA tetrahedral structure is added in 1mL 15nm gold size solution, 100mM PB buffer (pH7.4) is added after shaken at room temperature spends the night, final concentration is 10mM, Ageing solution (10 mM PB are added after ambient temperature overnight, 2M NaCl, pH 7.4) final concentration of salt is 0.15M in solution.After mixture solution ambient temperature overnight, 4 DEG C of 12000rpm/min, 15min, after centrifuge washing 3 times, throw out is in 200 μ L, pH7.4 PB(10 mM, 0.15 M NaCl) resuspension in solution, 4 DEG C of preservations.The Au-DNA tetrahedron AFM prepared carries out reclaiming, characterizing.
5 μ L 100 μMs preformation circular DNAs and 10 μ L 100 μMs of complementary sequences join in 0.5mL centrifuge tube and mix, 1min at 90 DEG C, when in pipe, solution naturally cools to room temperature, add 3 μ LT4 ligase enzymes and 2 μ LT4 ligase enzyme damping fluids, more than 16 hours are connected under 25 DEG C of environment, after terminating, enzyme denaturation 10min under 65 DEG C of conditions, then add 3 μ LT4 polysaccharases and 6 μ LT4 polymerase buffers, react more than 24 hours at 37 DEG C after solution is mixed.After enzyme effect completely, at high temperature (85 DEG C) process 10min, all sex change is complete for all enzymes making in solution.Finally use the super filter tube ultrafiltration (10000rpm/min, 5min) of 30KD, collecting bottom solution, is the mixing solutions containing cyclic DNA.
Following solution is added: circular DNA (6 μ L), dNTPs (2 μ L, 10 mM) in 8 μ L Au-DNA tetrahedrons, RCA buffer (2 μ L, 10 ×), phi29 polymerase (2 μ L, 10 U/ μ L), mixing solutions final volume is 20 μ L.Then increase in 30 DEG C of water-baths 30 min.Products therefrom 4 DEG C, the centrifugal 5min of 5000 rpm/min, carefully removes supernatant, after adding 20 μ L Milli-Q water resuspensions, gets 10 μ L loadings, 1% agarose electrophoresis, voltage 120V, 30min.Another 10 μ L are diluted in 90 μ LMilli-Q water, get 5 μ L and drip to smooth clean mica surface, and after absorption 3min, Milli-Q drip rinses, atomic force microscope Tapping-mode imaging.
Embodiment 5:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-HS-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-HS-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-HS-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.DNA tetrahedron PAGE and AFM prepared carries out reclaiming, characterizing.
20 μ L (10 μMs) DNA tetrahedral structure is added in 1mL 15nm gold size solution, 100mM PB buffer (pH7.4) is added after shaken at room temperature spends the night, final concentration is 10mM, Ageing solution (10 mM PB are added after ambient temperature overnight, 2M NaCl, pH 7.4) final concentration of salt is 0.15M in solution.After mixture solution ambient temperature overnight, 4 DEG C of 12000rpm/min, 15min, after centrifuge washing 3 times, throw out is in 200 μ L, pH7.4 PB(10 mM, 0.15 M NaCl) resuspension in solution, 4 DEG C of preservations.The Au-DNA tetrahedron AFM prepared carries out reclaiming, characterizing.
5 μ L 100 μMs preformation circular DNAs and 10 μ L 100 μMs of complementary sequences join in 0.5mL centrifuge tube and mix, 1min at 90 DEG C, when in pipe, solution naturally cools to room temperature, add 3 μ LT4 ligase enzymes and 2 μ LT4 ligase enzyme damping fluids, more than 16 hours are connected under 25 DEG C of environment, after terminating, enzyme denaturation 10min under 65 DEG C of conditions, then add 3 μ LT4 polysaccharases and 6 μ LT4 polymerase buffers, react more than 24 hours at 37 DEG C after solution is mixed.After enzyme effect completely, at high temperature (85 DEG C) process 10min, all sex change is complete for all enzymes making in solution.Finally use the super filter tube ultrafiltration (10000rpm/min, 5min) of 30KD, collecting bottom solution, is the mixing solutions containing cyclic DNA.
Following solution is added: circular DNA (6 μ L) after adding 8 μ LmilliQ water resuspensions in Au-DNA tetrahedron, bio-dNTPs (2 μ L, 10 mM), RCA buffer (2 μ L, 10 ×), phi29 polymerase (2 μ L, 10 U/ μ L), mixing solutions final volume is 20 μ L.Then increase in 30 ° of C water-baths 30 min.After products therefrom centrifuge washing three times, add 10 μ L 1/1000 avidin-HRP solution, in 37 DEG C of water-baths, hatch 30 min.After terminating, 200 μ L washingss (150mmol/L PBS, 0.25% Tween20) wash three times, add TMB(containing H
2o
2) solution 100 μ L, after after solution colour becomes basket, color no longer changes, solution absorption value in microplate reader detect aperture.
Embodiment 6:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-NH
2-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-NH
2-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-NH
2-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.DNA tetrahedron PAGE and AFM prepared carries out reclaiming, characterizing.20 μ L (10 μMs) DNA tetrahedral structure in 10 μ L magnetic beads solution (1mM), 37 DEG C of shaken overnight are placed on magnet stand, magnetic levitation, reject supernatant (the DNA tetrahedron do not connected), add 100 μ LpH7.4 PB(10 mM, 0.15 M NaCl) wash three times.Resuspension in 20 μ L PB buffer solution is added, 4 DEG C of preservations in throw out.
5 μ L 100 μMs preformation circular DNAs and 10 μ L 100 μMs of complementary sequences join in 0.5mL centrifuge tube and mix, 1min at 90 DEG C, when in pipe, solution naturally cools to room temperature, add 3 μ LT4 ligase enzymes and 2 μ LT4 ligase enzyme damping fluids, more than 16 hours are connected under 25 DEG C of environment, after terminating, enzyme denaturation 10min under 65 DEG C of conditions, then add 3 μ LT4 polysaccharases and 6 μ LT4 polymerase buffers, react more than 24 hours at 37 DEG C after solution is mixed.After enzyme effect completely, at high temperature (85 DEG C) process 10min, all sex change is complete for all enzymes making in solution.Finally use the super filter tube ultrafiltration (10000rpm/min, 5min) of 30KD, collecting bottom solution, is the mixing solutions containing cyclic DNA.
Following solution is added: circular DNA (6 μ L) after adding 8 μ LmilliQ water resuspensions in magnetic bead-DNA tetrahedron, bio-dNTPs (2 μ L, 10 mM), RCA buffer (2 μ L, 10 ×), phi29 polymerase (2 μ L, 10 U/ μ L), mixing solutions final volume is 20 μ L.Then increase in 30 DEG C of water-baths 30 min.After products therefrom magnetic levitation washs three times, add 10 μ L 1/1000 avidin-HRP solution, in 37 DEG C of water-baths, hatch 30 min.After terminating, 200 μ L washingss (150mmol/L PBS, 0.25% Tween20) wash three times, add TMB(containing H
2o
2) solution 100 μ L, after after solution colour becomes basket, color no longer changes, solution absorption value in microplate reader detect aperture.
Embodiment 7:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-NH
2-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-NH
2-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-NH
2-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.DNA tetrahedron PAGE and AFM prepared carries out reclaiming, characterizing.20 μ L (10 μMs) DNA tetrahedral structure in 10 μ L magnetic beads solution (1mM), 37 DEG C of shaken overnight are placed on magnet stand, magnetic levitation, reject supernatant (the DNA tetrahedron do not connected), add 100 μ LpH7.4 PB(10 mM, 0.15 M NaCl) wash three times.
5 μ L 100 μMs preformation circular DNAs and 10 μ L 100 μMs of complementary sequences join in 0.5mL centrifuge tube and mix, 1min at 90 DEG C, when in pipe, solution naturally cools to room temperature, add 3 μ LT4 ligase enzymes and 2 μ LT4 ligase enzyme damping fluids, more than 16 hours are connected under 25 DEG C of environment, after terminating, enzyme denaturation 10min under 65 DEG C of conditions, then add 3 μ LT4 polysaccharases and 6 μ LT4 polymerase buffers, react more than 24 hours at 37 DEG C after solution is mixed.After enzyme effect completely, at high temperature (85 DEG C) process 10min, all sex change is complete for all enzymes making in solution.Finally use the super filter tube ultrafiltration (10000rpm/min, 5min) of 30KD, collecting bottom solution, is the mixing solutions containing cyclic DNA.
Following solution is added: circular DNA (6 μ L) after adding 8 μ LmilliQ water resuspensions in magnetic bead-DNA tetrahedron, dNTPs (2 μ L, 10 mM), RCA buffer (2 μ L, 10 ×), phi29 polymerase (2 μ L, 10 U/ μ L), mixing solutions final volume is 20 μ L.Then increase in 30 ° of C water-baths 30 min.After products therefrom centrifuge washing three times, add the nutrient solution that 50 μ L contain some amount CTC, 200 μ L washings (150mmol/L PBS after 1h are hatched in 37 DEG C of incubators, 0.25% Tween20) wash three times, join in Tissue Culture Dish, after paraformaldehyde (or formaldehyde) fixing agent DAPI dyes, the imaging of confocal fluorescent basis of microscopic observation.
Embodiment 8:
The A chain of equivalent (
5 '-gTG AAAAA AAAAA GCA-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3 '), B chain (5'-SH-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'), C chain (5'-SH-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C-3'), D chain (5'-SH-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3') and TM buffer (20mM, Tris, 50mM MgCl
2, pH 8.0), fully after mixing, heat 2min at 95 DEG C, be then down to rapidly 4 DEG C in 30s.
20 μ L (10 μMs) DNA tetrahedral structure is added in 1mL 15nm gold size solution, 100mM PB buffer (pH7.4) is added after shaken at room temperature spends the night, final concentration is 10mM, Ageing solution (10 mM PB are added after ambient temperature overnight, 2M NaCl, pH 7.4) final concentration of salt is 0.15M in solution.After mixture solution ambient temperature overnight, 4 DEG C of 12000rpm/min, 15min, after centrifuge washing 3 times, throw out is in 200 μ L, pH7.4 PB(10 mM, 0.15 M NaCl) resuspension in solution, 4 DEG C of preservations.
5 μ L 100 μMs preformation circular DNAs and 10 μ L 100 μMs of complementary sequences join in 0.5mL centrifuge tube and mix, 1min at 90 DEG C, when in pipe, solution naturally cools to room temperature, add 3 μ LT4 ligase enzymes and 2 μ LT4 ligase enzyme damping fluids, more than 16 hours are connected under 25 DEG C of environment, after terminating, enzyme denaturation 10min under 65 DEG C of conditions, then add 3 μ LT4 polysaccharases and 6 μ LT4 polymerase buffers, react more than 24 hours at 37 DEG C after solution is mixed.After enzyme effect completely, at high temperature (85 DEG C) process 10min, all sex change is complete for all enzymes making in solution.Finally use the super filter tube ultrafiltration (10000rpm/min, 5min) of 30KD, collecting bottom solution, is the mixing solutions containing cyclic DNA.
Following solution is added: circular DNA (6 μ L) after adding 8 μ LmilliQ water resuspensions in Au-DNA tetrahedron, dNTPs (2 μ L, 10 mM), RCA buffer (2 μ L, 10 ×), phi29 polymerase (2 μ L, 10 U/ μ L), mixing solutions final volume is 20 μ L.Then increase in 30 ° of C water-baths 30 min.After products therefrom centrifuge washing three times, add the nutrient solution that 50 μ L contain some amount CTC, 200 μ L washings (150mmol/L PBS after 1h are hatched in 37 DEG C of incubators, 0.25% Tween20) wash three times, join in Tissue Culture Dish, after paraformaldehyde (or formaldehyde) fixing agent DAPI dyes, the imaging of confocal fluorescent basis of microscopic observation.
Claims (8)
1. a nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction, it is characterized in that, after the DNA tetrahedron prepared is assembled in nanoparticle surface, one section of single stranded DNA that DNA tetrahedron extends can be used as primed DNA, use with the annular DNA of specific aptamer complementary as amplification template, hybridize with primed DNA and cause rolling circle amplification and react, the product of preparation is many long single stranded DNAs of nanoparticle surface many apatmer structure composition, be used as cell detection by the combination of aptamer and cell surface special receptor, catch and cell imaging probe.
2. according to claim 1 nanoparticle surface DNA tetrahedron be support and cause rolling circle amplification reaction method, it is characterized in that, described DNA tetrahedron be four single stranded DNAs by DNA hybridization, a kind of tetrahedral structure of self-assembly.
3. nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction according to claim 1, and it is characterized in that, described nanoparticle is the one in nanometer gold, quantum dot, magnetic nano-particle.
4. nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction according to claim 1, it is characterized in that, described DNA tetrahedron be assembled in nanoparticle surface be the DNA tetrahedron of sulfydryl modification by Au-S key, or NH
2the DNA tetrahedron of-modification is assembled in nanometer gold surface by condensation reaction etc. or has the nanoparticle surface containing-COOH base.
5. nanoparticle surface DNA tetrahedron according to claim 1 is support and causes the method for rolling circle amplification reaction, it is characterized in that one section of single stranded DNA of described extension is the section of DNA sequence of 10-30 base.
6. nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction according to claim 1, it is characterized in that, described specific aptamer sequence be acceptor deposit in case can with the section of DNA sequence of its specific binding, as the aptamer structure that can combine with circulating tumor cell surface receptor (EpCAM).
7. nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction according to claim 1, it is characterized in that, described amplification template is the DNA unrelated sequences containing one section of non-aptamer complementation.
8. nanoparticle surface DNA tetrahedron is support and causes the method for rolling circle amplification reaction according to claim 1, it is characterized in that, many long single stranded DNAs of described many apatmer structure composition are the DNA single chain that aptamer structure nothing to do with train interval occurs, unrelated sequences can reduce the steric effect of aptamer and receptors bind.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN106754894A (en) * | 2017-02-28 | 2017-05-31 | 临沂大学 | A kind of multifunction magnetic DNA nanospheres and preparation method and application |
| CN108085369A (en) * | 2017-12-28 | 2018-05-29 | 东南大学 | A kind of method of-four serobila DNA of quantum dot fluorescence coding microball-rolling circle amplification high throughout screening of G |
| CN109187468A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | It is a kind of to mediate nitrating carbon dots to be catalyzed H with aptamers2O2The method of fluorescence spectrum method for measuring isocarbophos is reacted with TMB |
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| CN111019941A (en) * | 2020-01-21 | 2020-04-17 | 福州大学 | DNA nano material and preparation method and application thereof |
| CN111172246A (en) * | 2020-01-13 | 2020-05-19 | 天津国科医工科技发展有限公司 | Electrochemical nucleic acid detection method based on DNA walking and rolling circle amplification signal amplification |
| CN111638330A (en) * | 2020-06-10 | 2020-09-08 | 青岛农业大学 | Biosensor for detecting salmonella typhimurium and application thereof |
| CN111974985A (en) * | 2020-09-16 | 2020-11-24 | 南京大学 | Nano particle cluster assembling method using micro magnetic beads as growth template and DNA frame as guide carrier |
| CN112439369A (en) * | 2021-02-01 | 2021-03-05 | 中国农业大学 | Preparation method of DNA regular tetrahedron-rolling circle amplification product double-crosslinked hydrogel |
| CN112924695A (en) * | 2021-01-19 | 2021-06-08 | 中国计量科学研究院 | Composite magnetic nano material based on DNA tetrahedron, preparation and application |
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| CN109517722B (en) * | 2018-09-28 | 2022-04-01 | 德拜至臻医学科技(杭州)有限公司 | Device for capturing specific trace cells and manufacturing and using methods thereof |
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| CN111019941A (en) * | 2020-01-21 | 2020-04-17 | 福州大学 | DNA nano material and preparation method and application thereof |
| CN111019941B (en) * | 2020-01-21 | 2023-09-08 | 福州大学 | DNA nano material and preparation method and application thereof |
| CN111638330A (en) * | 2020-06-10 | 2020-09-08 | 青岛农业大学 | Biosensor for detecting salmonella typhimurium and application thereof |
| CN111638330B (en) * | 2020-06-10 | 2021-04-02 | 青岛农业大学 | Biosensor for detecting salmonella typhimurium and application thereof |
| CN111974985B (en) * | 2020-09-16 | 2022-03-01 | 南京大学 | Nano particle cluster assembling method using micro magnetic beads as growth template and DNA frame as guide carrier |
| CN111974985A (en) * | 2020-09-16 | 2020-11-24 | 南京大学 | Nano particle cluster assembling method using micro magnetic beads as growth template and DNA frame as guide carrier |
| CN112924695A (en) * | 2021-01-19 | 2021-06-08 | 中国计量科学研究院 | Composite magnetic nano material based on DNA tetrahedron, preparation and application |
| CN112439369A (en) * | 2021-02-01 | 2021-03-05 | 中国农业大学 | Preparation method of DNA regular tetrahedron-rolling circle amplification product double-crosslinked hydrogel |
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