CN101368198A - Preparation method for self-assembly material of nano golden and Lambda DNA chain connection - Google Patents
Preparation method for self-assembly material of nano golden and Lambda DNA chain connection Download PDFInfo
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- CN101368198A CN101368198A CNA200810156724XA CN200810156724A CN101368198A CN 101368198 A CN101368198 A CN 101368198A CN A200810156724X A CNA200810156724X A CN A200810156724XA CN 200810156724 A CN200810156724 A CN 200810156724A CN 101368198 A CN101368198 A CN 101368198A
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Abstract
The invention discloses a preparation method of a self-assembled material, in which connected by nanometer gold and a Lambda DNA chain are connected. The preparation method of the self-assembled material belongs to the technical field of material chemistry. The main content of the invention is PCR on a gold nanometer particle interface; the steps are as follows: (1) preparing colloidal gold; (2), coupling the colloidal gold with upstream and downstream primers; (3) carrying out PCR on the prepared primers connected with gold and carrying out agarose gel electrophoresis and TEM scanning on the output for proving to obtain an expected output; firstly preparing the AuNPS of needed grain diameters and respectively connecting the AuNPS with the upstream and downstream primers decorated by-SH to form the primer compound of the gold nanometer grains; then carrying out PCR by taking the Lambda DNA as a template and utilizing the prepared primer compound of the gold nanometer grains to obtain the self-assembled material connected by the AuNPS and the Lambda DNA chain. As a DNA structure has the characters of melting at a high temperature and renaturation at a lower temperature which are complementary and in pair with a base, the novel nanometer mateirla becomes a self-assembled material with contorlable temperautre and has applicatoin potentials in a plurality of fields.
Description
Technical field
The preparation method of the self-assembled material that a kind of nanometer gold is connected with λ DNA chain belongs to material chemistry technical field.
Background technology
Along with the continuous infiltration of nanotechnology to life science, utilize nanometer biotechnology research and solution significant problem wherein, promote the development of nanometer biotechnology, just becoming when one of previous important advanced field.Gelationus golden nanometer particle (AuNPs) is because its special physics, chemical property and biocompatibility have application very widely in fields such as bioelectrochemistry, materialogy and biomedicines.
Polymerase chain reaction (PCR) is a kind of external specific nucleic acid sequence amplification technique, is one of cut-and-try work basis of modern molecular biology, is bringing into play important effect in fields such as life science, genetics and medical science.PCR carries out in the solution of homogeneous usually, and can carry out PCR on solid interface smoothly be a problem with exploration.Recently, people such as Turner utilizes PCR on micropore surface to detect the sudden change of hemochrome gene; People such as Adessi have studied the PCR of glass surface, have inquired into the possibility of development DNA chip; People such as Lockley have done preliminary exploration to the PCR on the nylon surface.PCR on the nanoparticle interface yet there are no report.If on the nanoparticle interface, can carry out PCR smoothly, can widen the range of application of round pcr greatly, the round pcr in the homogeneous system is expanded on the nanoparticle interface.Therefore primer is connected on the nanoparticle interface, the PCR on the research nanoparticle interface is significant.Systematic study of the present invention PCR on the golden nanometer particle interface.Be connected produced on the nanoparticle interface sterically hindered in order to reduce primer, connection-(CH between nanoparticle and primer
2)
6-as the buffering connecting arm.By the Au-S key 5 ＇ terminal modified-SH-(CH
2)
6-primer be connected the golden nanometer particle surface, adopt plasmid λ DNA as template, with agarose electrophoresis and transmission electron microscope technology such as (TEM) the PCR product is analyzed and detected, studied the influence of primer concentration, annealing temperature and cycle index PCR.Experimental result shows: two kinds of primers of R, F are connected on the golden nanometer particle interface, can carry out PCR smoothly, and golden nanometer particle can be connected to form the nanoparticle polymer, low temperature renaturation and base complementrity paired character because dna structure high temperature unwinds make this novel nano-material become a kind of self-assembled material with Controllable Temperature.
Summary of the invention
The purpose of this invention is to provide the preparation method of the self-assembled material that a kind of nanometer gold is connected with λ DNA chain, this kind novel nano self-assembled material has self-assembly and Controllable Temperature simultaneously.
Technical scheme of the present invention: the preparation method of the self-assembled material that a kind of nanometer gold is connected with λ DNA chain, step is: the preparation of (1) Radioactive colloidal gold; (2) coupling of Radioactive colloidal gold and upstream and downstream primer; (3) utilize the golden primer of prepared company to carry out PCR, the PCR product carries out agarose electrophoresis and TEM electron-microscope scanning proof and obtains expecting product;
(1) preparation of Radioactive colloidal gold: utilize the Frens legal system to be equipped with Radioactive colloidal gold about 15nm.
Used glassware all strong acid soaks, and distilled water cleans, and oven for drying is standby; During preparation, adding 100mL mass concentration is 0.01% hydrochloro-auric acid and clean stirrer in the Erlenmeyer flask of cleaning, middling speed stirs, and is heated to boiling, keeps 5～6 minutes, and then disposable adding mass concentration is 1% citric acid three sodium solution 4mL, heat while stirring, solution colour is from faint yellow light blue black, the extremely dark blue black of becoming successively, become burgundy at last, after color no longer changes, continue heating again and reacted fully in 15 minutes fully; At last, under stirring condition, solution is cooled to room temperature, and constant volume is to 100mL, 4 ℃ of preservations;
(2) coupling of Radioactive colloidal gold and upstream and downstream primer: prepared Radioactive colloidal gold is connected respectively with primers F, R, golden nanometer particle-primer complex, i.e. AuNP-F and AuNP-R, it is standby to handle the back.
Used upstream and downstream primer (it is synthetic to give birth to the worker by Shanghai) is:
297bp-F：5’-GCAGTTGCCGTTTATCTCACC-3’，
297bp-R：5’-GGCATAGCGTCCTCACATTTC-3’，
With prepared Radioactive colloidal gold in the centrifugal 15min of 11500rpm, abandon supernatant, return to 1/10 of original volume with aqua sterilisa, to go up, the downstream primer concentration dilution is to 10um, with Radioactive colloidal gold: upstream and downstream primer R or 1: 1 ratio of F volume ratio are mixed respectively, about ultrasonic 10s, make it to mix, room temperature leaves standstill 12h, to the NaCl that wherein adds 1M, make the NaCl concentration in the system reach 0.05M, continue room temperature and leave standstill 12h, reaction finishes, with the centrifugal 15min of 6500rpm, abandon supernatant earlier, add the phosphate buffer soln eccentric cleaning that contains 0.1mol/LNaCl pH 7.0 then, repeated washing is repeatedly removed the primer that is not connected on the golden nanometer particle interface, it is stand-by to be settled to the volume of Radioactive colloidal gold with aqua sterilisa at last, and primer is connected the golden nanometer particle surface, constitutes golden nanometer particle-primer complex, be AuNP-F and AuNP-R, 4 ℃ of preservations are standby;
(3) PCR: with λ DNA is template, adopts the primer AuNP-F and the AuNP-R that make to carry out PCR.PCR reaction system: dNTPs (giving birth to the worker) 1 μ L available from Shanghai, 0.5 the Taq archaeal dna polymerase of individual unit (giving birth to the worker) available from Shanghai, λ dna profiling plasmid (available from precious biotechnology company limited) 0.5 μ L, reaction buffer (giving birth to the worker) 5 μ L available from Shanghai, get AuNP-F 3 μ L and AuNP-R 3 μ L, adding aqua sterilisa to cumulative volume is 50 μ L;
Amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 40 circulations; 72 ℃ are extended 3min again; 10 ℃ of preservations get the PCR product;
(4) agarose electrophoresis: get 5 μ LPCR products, analyze with 3% agarose gel electrophoresis, dye before the employing, promptly add bromination second pyridine (EB) in earlier stage at colloidal sol, concentration is about 0.5 μ g/mL, voltage U=120V, and time T=30min carries out electrophoresis;
(5) TEM electron-microscope scanning
The PCR product that obtains is added drop-wise on the copper mesh with liquid-transfering gun, places under the lamp oven dry half an hour, last TEM Electronic Speculum finds suitable structure and magnification and takes pictures.
Description of drawings
The TEM figure of Fig. 1 15nm gold.
The electrophorogram that is connected of Fig. 2 Radioactive colloidal gold and upstream and downstream primer.
The electrophorogram of Fig. 3 PCR product.
The TEM figure of Fig. 4 PCR product.
Embodiment
(1) preparation of Radioactive colloidal gold:
Used glassware all strong acid soaks, and distilled water cleans, and oven for drying is standby.During preparation, adding 100mL mass concentration is 0.01% hydrochloro-auric acid and clean stirrer in the Erlenmeyer flask of cleaning, middling speed stirs, be heated to boiling, kept 5～6 minutes, and then disposable adding mass concentration is 1% citric acid three sodium solution 4mL, heat while stirring, solution colour becomes light blue black successively from faint yellow, becomes burgundy at last to dark blue black, continues heating again and reacted fully in 15 minutes fully after color no longer changes.At last, under stirring condition, solution is cooled to room temperature, and constant volume is to 100mL, 4 ℃ of preservations.
(2) coupling of Radioactive colloidal gold and upstream and downstream primer:
Used upstream and downstream primer is:
297bp-F：5’-GCAGTTGCCGTTTATCTCACC-3’，
297bp-R：5’-GGCATAGCGTCCTCACATTTC-3’，
With the centrifugal 15min of prepared Radioactive colloidal gold 11500rpm, abandon supernatant, aqua sterilisa returns to 1/10 of original volume, the upstream and downstream primer concentration is diluted to 10um, with Radioactive colloidal gold: 1: 1 ratio of upstream and downstream primer (R or F) volume ratio is mixed respectively, about ultrasonic 10s, make it to mix, room temperature leaves standstill 12h, to the NaCl that wherein adds 1M, NaCl concentration in the last system reaches 0.05M, continue room temperature and leave standstill 12h, reaction finishes, earlier with the centrifugal 15min of 6500rpm, abandon supernatant, add phosphoric acid salt buffered soln (containing 0.1mol/LNaCl pH 7.0) eccentric cleaning then.Remove the primer that is not connected on the golden nanometer particle interface with above-mentioned method of repeatedly cleaning.Stand-by with the volume that is settled to Radioactive colloidal gold in the aqua sterilisa at last.Primer is connected nanoparticle surface, constitutes golden nanometer particle-primer complex, i.e. AuNP-F and AuNP-R, 4 ℃ of preservations are standby.
(3) PCR: with λ DNA is template, adopts the primer AuNP-F and the AuNP-R that make to carry out PCR.
The PCR reaction system comprises 1 μ L dNTPs, the Taq archaeal dna polymerase of 0.5 unit, and 0.5 μ L λ dna profiling plasmid, 5 μ L reaction buffers are got AuNP-F 3 μ L and AuNP-R 3 μ L, and adding aqua sterilisa to cumulative volume at last is 50 μ L.
Amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 40 circulations; 72 ℃ are extended 3min again.10 ℃ of preservations.
(4) agarose electrophoresis
Get 5 μ L PCR products, analyze with 3% agarose gel electrophoresis.Dye before the employing, promptly add EB in earlier stage at colloidal sol, concentration is about 0.5ug/mL, voltage U=120V, time T=30min.Carry out electrophoresis.
(5) TEM electron-microscope scanning
The PCR product that obtains is added drop-wise on the copper mesh with liquid-transfering gun, places under the lamp oven dry, approximately about half an hour, last TEM Electronic Speculum finds suitable structure and magnification and takes pictures.
Claims (1)
1. the preparation method of the self-assembled material that is connected with λ DNA chain of a nanometer gold is characterized in that step is: the preparation of (1) Radioactive colloidal gold; (2) coupling of Radioactive colloidal gold and upstream and downstream primer; (3) utilize the golden primer of prepared company to carry out PCR, the PCR product carries out agarose electrophoresis and TEM electron-microscope scanning proof and obtains expecting product;
(1) preparation of Radioactive colloidal gold: utilize the Frens legal system to be equipped with the Radioactive colloidal gold of 15nm;
Used glassware all strong acid soaks, and distilled water cleans, and oven for drying is standby; During preparation, adding 100mL mass concentration is 0.01% hydrochloro-auric acid and clean stirrer stirring in the Erlenmeyer flask of cleaning, be heated to boiling, kept 5～6 minutes, and then disposable adding mass concentration is 1% citric acid three sodium solution 4mL, heats while stirring, solution colour becomes light blue black successively from faint yellow, to dark blue black, become burgundy at last, after color no longer changes, continue heating again and reacted fully in 15 minutes fully; At last, under stirring condition, solution is cooled to room temperature, and constant volume is to 100mL, 4 ℃ of preservations;
(2) coupling of Radioactive colloidal gold and upstream and downstream primer: prepared Radioactive colloidal gold is connected respectively with primers F, R, golden nanometer particle-primer complex, i.e. AuNP-F and AuNP-R, it is standby to handle the back;
Used upstream and downstream primer is:
297bp-F：5’-GCAGTTGCCGTTTATCTCACC-3’，
297bp-R：5’-GGCATAGCGTCCTCACATTTC-3’，
With prepared Radioactive colloidal gold in the centrifugal 15min of 11500rpm, abandon supernatant, return to 1/10 of original volume with aqua sterilisa, to go up, the downstream primer concentration dilution is to 10um, with Radioactive colloidal gold: upstream and downstream primer R or 1: 1 ratio of F volume ratio are mixed respectively, and ultrasonic 10s makes it to mix, room temperature leaves standstill 12h, to the NaCl that wherein adds 1M, make the NaCl concentration in the system reach 0.05M, continue room temperature and leave standstill 12h, reaction finishes, with the centrifugal 15min of 6500rpm, abandon supernatant earlier, add the phosphate buffer soln eccentric cleaning that contains 0.1mol/L NaCl pH 7.0 then, repeated washing is repeatedly removed the primer that is not connected on the golden nanometer particle interface, it is stand-by to be settled to the volume of Radioactive colloidal gold with aqua sterilisa at last, and primer is connected the golden nanometer particle surface, constitutes golden nanometer particle-primer complex, be AuNP-F and AuNP-R, 4 ℃ of preservations are standby;
(3) PCR: with λ DNA is template, adopts the primer AuNP-F and the AuNP-R that make to carry out PCR;
PCR reaction system: dNTPs 1 μ L, the Taq archaeal dna polymerase of 0.5 unit, λ dna profiling plasmid 0.5 μ L, reaction buffer 5 μ L get AuNP-F 3 μ L and AuNP-R 3 μ L, and adding aqua sterilisa to cumulative volume is 50 μ L;
Amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 40 circulations; 72 ℃ are extended 3min again; 10 ℃ of preservations get the PCR product;
(4) agarose electrophoresis:
Get 5 μ L PCR products, analyze with 3% agarose gel electrophoresis, dye before the employing, promptly add the pyridine of bromination second in earlier stage at colloidal sol, concentration is 0.5 μ g/mL, voltage U=120V, and time T=30min carries out electrophoresis;
(5) TEM electron-microscope scanning.
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