CN109182480B - Application of metal organic framework material in polymerase chain reaction - Google Patents
Application of metal organic framework material in polymerase chain reaction Download PDFInfo
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Abstract
The invention discloses an application of a metal organic framework material in polymerase chain reaction, belonging to the field of molecular biology. The application comprises the step of adding a metal organic framework material suspension into a polymerase chain reaction system for nucleic acid amplification, wherein the ratio of the mass of the metal organic framework material to the volume of the polymerase chain reaction system is 10mg/L-100 mg/L. The polymerase chain reaction is conventional PCR, nested PCR or multiplex PCR. The application of the metal organic framework material in PCR can obviously improve the specificity, sensitivity and amplification yield of PCR, is simple and convenient to use, has excellent comprehensive optimization effect, and has wide application value.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for optimizing polymerase chain reaction by using a metal organic framework material.
Background
The metal organic framework material is a novel organic-inorganic complex porous crystal material, is formed by taking a metal center or an inorganic cluster as a node and taking carboxylic acid or nitrogen-containing organic polydentate ligand as a skeleton through coordination and self-assembly, and has a periodic multidimensional regular pore channel structure, an ultrahigh specific surface area and a permanent porosity. Because the preparation method is simple, regular and ordered, the aperture and the shape can be adjusted, and the functionalization is easy, the metal organic framework material is widely applied to a plurality of fields such as gas adsorption, separation and purification, catalysis, degradation, biological sensing, medicine loading and the like as a multifunctional material. UiO-66 and ZIF-8 are two more studied metal organic framework materials, both of which haveHas excellent hydrothermal and chemical stability, wherein UiO-66 is a Zr-centered, terephthalic acid (H)2BDC) is a rigid metal organic framework material of an organic ligand; ZIF-8 is a metal organic framework material with a zeolite sodalite topology synthesized from divalent zinc salt and 2-methylimidazole.
The polymerase chain reaction is an in vitro nucleic acid amplification technique, which uses trace genes from biological samples such as blood, hair, skin, saliva, etc. as templates, and repeatedly performs melting-annealing-extension steps to increase the target genes by millions of times within hours. Although the technology is mature at present, due to the lack of precise regulation and mismatch repair mechanisms similar to in vivo amplification, non-ideal amplification results such as few amplification products, non-specific amplification, amplification failure and the like often occur. In recent years, nanoparticles (such as gold nanoparticles, carbon nanopowders, quantum dots, etc.) have been introduced into PCR amplification systems as a special additive, and nonspecific amplification, yield, and sensitivity of PCR have been improved to various degrees. However, the reported nanoparticles have a strong emphasis on improving PCR, and often cannot improve specificity, yield and sensitivity at the same time, so that the comprehensive optimization efficiency is not ideal.
Disclosure of Invention
The invention solves the technical problems of increased nonspecific amplification, low yield and low sensitivity of PCR reaction in the prior art, and provides the application of the metal organic framework material in polymerase chain reaction.
According to the object of the present invention, the use of a metal-organic framework material in polymerase chain reactions is provided.
Preferably, the metal organic framework material is a three-dimensional porous material formed by coordination of zirconium ions and dicarboxylic acid ligands or a zeolite-like imidazolate framework compound.
Preferably, the metal-organic framework material is a metal-organic framework material which takes zirconium ions as a metal center and takes terephthalic acid as an organic ligand; or the metal organic framework material takes zinc ions as a metal center and takes 2-methylimidazole as an organic ligand.
Preferably, the application is that the metal organic framework material suspension is added into a polymerase chain reaction system for nucleic acid amplification.
Preferably, the ratio of the mass of the metal organic framework material to the volume of the polymerase chain reaction system is 10mg/L-100 mg/L.
Preferably, the template DNA amplified by the polymerase chain reaction is lambda phage DNA, plasmid DNA, mRNA, animal genomic DNA, plant genomic DNA, or human genomic DNA.
Preferably, the lambda phage DNA template concentration is greater than or equal to 0.13. mu.g/L.
Preferably, the polymerase chain reaction is a conventional polymerase chain reaction, a reverse transcription polymerase chain reaction, a nested polymerase chain reaction or a multiplex polymerase chain reaction.
Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) as a new achievement of combining the metal organic framework material and biotechnology, the metal organic framework material has nanometer characteristics of ultrahigh specific surface area, biomolecule adsorption and the like, the specificity of PCR reaction can be obviously enhanced after the metal organic framework material is added into a PCR reaction system, the sensitivity of the reaction is improved by at least 8 times, the PCR yield is improved by about 5 times under the template concentration capable of generating stable strips, and the comprehensive optimization effect is better.
(2) The invention provides a new application of the metal organic framework material, and simultaneously provides a new additive for improving the PCR effect. The application has obvious optimization effect on PCR, low application cost, simple and convenient use and easy grasp.
(3) The metal organic framework material used in the invention has the advantages of simple preparation steps, good water solubility and convenient storage.
Drawings
FIG. 1 is a representation of nanoparticles in a UiO-66 suspension prepared according to the present invention; wherein: FIG. 1(a) is a PXRD (polycrystalline powder X-ray diffraction) characterization chart of UiO-66 after synthesis; FIG. 1(b) is a nitrogen adsorption-desorption diagram of BET specific surface area; FIG. 1(c) is an SEM scanning electron micrograph of UiO-66 particles after sonication and sterilization.
FIG. 2 is a photograph of a characterization of nanoparticles in a ZIF-8 suspension prepared according to the present invention; wherein: FIG. 2(a) is a representation of PXRD (polycrystalline powder X-ray diffraction) after ZIF-8 synthesis; FIG. 2(b) is a drawing showing adsorption-desorption of nitrogen at BET specific surface area; FIG. 2(c) is an SEM scanning electron micrograph of ZIF-8 particles after sonication and sterilization.
FIG. 3 is a graph showing the effect of UiO-66 and ZIF-8 on the specificity of the second round of PCR; wherein FIG. 3(a) is a graph showing the results of the first and second rounds of PCR; FIG. 3(b) is a graph showing the effect of different concentrations of UiO-66 on PCR specificity; FIG. 3(c) is a graph showing the effect of different concentrations of ZIF-8 on PCR specificity, lane M is a DNA molecular weight standard, lane 1 is a negative control, lanes 2-7 correspond to final concentrations of ZIF-8 of 1000,100,20,10,2,1mg/L, respectively, and lane 8 is a control without ZIF-8; FIG. 3(d) is a graph showing the results of PCR without the addition of metal organic framework material (lanes 1,2) and with the addition of optimal concentrations (20mg/L) of UiO-66 (lanes 3,4) or ZIF-8 (lanes 5, 6).
FIG. 4(a) is a graph of the effect of optimized UiO-66 and ZIF-8 concentrations on the sensitivity and yield of the first round of PCR, where lane M is the DNA molecular weight standard, the final concentrations of UiO-6 and ZIF-8 are 10mg/L, and lanes 1-7 are 0.125,0.25,0.5,1,2,4 and 20 μ g/L for λ -DNA template concentration, respectively; FIG. 4(b) is a graph showing the relative ratio of the gray scale of the bands of FIG. 4(a) in terms of the yield in terms of the double ratio at the same concentration.
FIG. 5 is a graph showing the effect of UiO-6 and ZIF-8 on the fluorescence intensity of a fluorescent single-stranded DNA fragment before and after addition of a complementary strand, wherein FP is a fluorescent single-stranded nucleic acid fragment and CP is a completely complementary nucleic acid fragment thereof.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1: preparation of UiO-66 (metal organic framework material with zirconium ion as metal center and terephthalic acid as organic ligand)
The preparation method and structural information characterization of UiO-66 are described in the literature (A New Zirconium Organic Building Brick Forming Metal Organic, J.Am.chem.Soc.2008,130(42),13850), specifically, ZrCl is firstly prepared4And 1, 4-benzanedicarboxylic acid (H)2BDC) was dissolved in DMF (molar ratio 1:1500, and the mixture was sealed and placed in an oven preheated to 120 ℃ for reaction for 24 hours. After the reaction mass is cooled to room temperature, it is activated by filtration, repeated DMF immersion and drying at room temperature. FIG. 1(a) is a PXRD (polycrystalline powder X-ray diffraction) characterization chart of UiO-66 after synthesis; FIG. 1(b) is a nitrogen adsorption-desorption diagram of BET specific surface area; FIG. 1(c) is an SEM scanning electron micrograph of UiO-66 particles after sonication and sterilization. FIG. 1(a) shows that the simulated peak of the product is consistent with the actual peak of the product, demonstrating the success of the synthesis. Fig. 1(b) shows a nitrogen adsorption and desorption graph of the material, and illustrates that the product is a mesoporous and microporous material. FIG. 1(c) demonstrates the characteristics of particle size and surface morphology of the particles in the suspension.
Example 2: preparation of ZIF-8 (metal organic framework material with zinc ion as metal center and 2-methylimidazole as organic ligand)
Preparation method and structural information characterization of ZIF-8 are described in literature (Aqueous room temperature synthesis of cobalt and zinc sol Zeolite framework, Dalton Trans.41,2012,5458), and raw material molar ratio is as follows: 2-methylimidazole: zn (NO)3)2·6H2O: deionized water 1:8: 1000. The specific experimental steps are as follows: (1) completely dissolving 2-methylimidazole in a proper amount of deionized water, and adjusting the pH value to 9.5 by using TEA buffer solution after complete dissolution; (2) adding Zn (NO)3)2·6H2Fully dissolving O in a small amount of deionized water; (3) completely mixing the solutions formed in the steps (1) and (2), and stirring to react for 20 minutes; (4) and pumping the stirred slurry into a microporous filter membrane filtering device by using an air compressor for suction filtration. In order to accelerate the suction filtration speed, nitrogen can be used for pressurization. (5) Washing with deionized water and anhydrous ethanol for 2-3 times respectively after filtering, and drying in a dryer;(6) and (3) activation: and dissolving the dried product in deionized water again, performing ultrasonic treatment for 3 times in an ultrasonic water bath for 2 hours, and performing vacuum drying for 3 days at the temperature of 120 ℃ for adjusting the price to remove excessive impurities such as 2-methylimidazole, water and the like in the product. FIG. 2 is a photograph of a characterization of nanoparticles in a ZIF-8 suspension prepared according to the present invention; wherein: FIG. 2(a) is a representation of PXRD (polycrystalline powder X-ray diffraction) after ZIF-8 synthesis; FIG. 2(b) is a drawing showing adsorption-desorption of nitrogen at BET specific surface area; FIG. 2(c) is an SEM scanning electron micrograph of ZIF-8 particles after sonication and sterilization. FIG. 2(a) shows that the simulated peak of the product is consistent with the actual peak of the product, demonstrating the success of the synthesis. Fig. 2(b) shows a nitrogen adsorption and desorption curve chart of the material, and the product is the mesoporous material. FIG. 2(c) shows particle size and surface morphology information of ZIF-8 particles used in the experiment.
Example 3: preparation of UiO-66/ZIF-8 pollution-free solution
Taking a prepared suspension solution with the concentration of 10mg/ml (W/V) as an example, 50mg of UiO-66/ZIF-8 powder obtained in the examples 1 and 2 is accurately weighed and placed in a sterile 15ml centrifuge tube, 5ml of ultrapure water is added by a pipette, a centrifuge tube cover is covered tightly, and then ultrasonic treatment is further carried out, wherein the specific ultrasonic power and ultrasonic time are subject to the conditions that UiO-66 particles are completely suspended and do not generate a layering phenomenon after standing at room temperature for at least 10 min. After the preparation of the stable suspension, the centrifuge tube was placed in an autoclave and sterilized at 121 ℃ for 30 min.
Example 4: optimization of PCR specificity by UiO-66/ZIF-8
The primer sequences used were as follows:
primer 1: 5'-GGCTTCGGTCCCTTCTGT-3', respectively;
primer 2: 5'-CACCACCTGTTCAAACTCTGC-3'
The PCR solution system composition (20. mu.L) was as follows: taq PCR mix 10. mu.L, primer 1 (10. mu.M) and primer 2 (10. mu.M) each 0.4. mu.L, template DNA 2. mu.L, different concentrations UiO-662. mu.L, sterile water 5.2. mu.L, total volume 20. mu.L.
The PCR procedure and conditions were as follows: (1) preheating at 95 ℃ for 5 min; (2) denaturation at 94 ℃ for 20 s; (3) annealing at 50 ℃ for 30 s; (4) extension at 72 ℃ for 30 s; extension at 72 ℃ for 5 min. Steps (2) - (4) were repeated for 35 cycles.
In order to clearly react the influence of UiO-66/ZIF-8 on the specificity of PCR, 283bp of target fragment of lambda phage DNA is firstly amplified, and then a second round of amplification is carried out by taking a 100-fold diluted product of a solution after reaction as a template. Wherein FIG. 3(a) is a graph showing the results of the first and second PCR cycles, lane M is a DNA molecular weight standard, lane 1 is the result of the second PCR cycle, and lane 2 is the result of the first PCR cycle; FIG. 3(b) is a graph showing the effect of different concentrations of UiO-66 on PCR specificity, lane M is a DNA molecular weight standard, lane 1 is a negative control, lanes 2-6 correspond to UiO-66 final concentrations of 1000,100,20,10,2mg/L, respectively, and lane 7 is a control without UiO-66; FIG. 3(c) is a graph showing the effect of different concentrations of ZIF-8 on PCR specificity, lane M is a DNA molecular weight standard, lane 1 is a negative control, lanes 2-7 correspond to final concentrations of ZIF-8 of 1000,100,20,10,2,1mg/L, respectively, and lane 8 is a control without ZIF-8. As shown in FIG. 3(a), the first round of amplification produced a bright single-purpose band, whereas the second round produced a diffuse, trailing band, indicating that PCR exhibited significant non-specific amplification. FIG. 3(b) compares the effect of adding no UiO-66 and different amount of UiO-66, and it can be seen from the figure that, compared with the experimental group without UiO-66, the tailing phenomenon is less and less as the concentration of UiO-66 in the system is increased, which indicates that the UiO-66 concentration can significantly improve the specificity of PCR amplification at a proper concentration. FIG. 3(c) compares the effect of adding no ZIF-8 and different amounts of ZIF-8, and it can be seen from FIG. 3(c) that the tailing phenomenon is less and less with the increase of the concentration of ZIF-8 in the system, compared with the experimental group without ZIF-8, indicating that the specificity of PCR amplification can be significantly improved by ZIF-8 at a proper concentration. FIG. 3(d) further compares the amplification results of the metal-organic framework material without addition of the metal-organic framework material and with addition of the metal-organic framework material at the optimum concentration, and more clearly demonstrates that UiO-66 and ZIF-8 significantly improve the PCR amplification specificity and the product yield.
Example 5: optimization of PCR sensitivity and yield using UiO-66 and ZIF-8
In order to clearly see the effect of UiO-66/ZIF-8 on PCR sensitivity, the first round of PCR was selected in this example. The experiment is divided into three groups, one group is not added with metal organic framework materials, the other group is added with 10mg/L UiO-66 in the PCR solution, and the third group is added with 10mg/L ZIF-8 in the PCR solution. 7 sets of experiments were performed in each set of systems, with 20,4,2,1,0.5,0.25 and 0.13. mu.g/L of lambda-DNA template, and the remaining components of the PCR solution system and PCR steps and conditions were the same as in example 4. The results are shown in FIG. 4, wherein lane M is DNA molecular weight standard, UiO-6 concentration is 10mg/L, and lanes 1-7 correspond to lambda-DNA template concentrations of 0.125,0.25,0.5,1,2,4, 20. mu.g/L, respectively. In FIG. 4, the group of experiments with ZIF-8 added is on the left, the group of experiments with UiO-66 added is in the middle, and the control group without MOFs is on the right. As can be seen from FIG. 4, with the increase of template concentration, the stable band could be seen only when the template concentration reached 1. mu.g/L in the experimental group without UiO-66, but the amplification product could still be stably detected at 0.13. mu.g/L in the experimental group with UiO-66, and the brightness of the band was higher than that of the 2. mu.g/L template product in the experimental group without UiO-66, indicating that the sensitivity of PCR was at least 8 times higher than that of conventional PCR. When the gray level of the bands in the agarose gel is measured by ImageJ software, the intensity is higher than that of the bands without adding the UiO-66, and the gray level ratio reaches about 5 times under the stably expressed template concentration of 1 mu g/L, namely the yield is increased by 4.7 times. ZIF-8 gave similar results to UiO-66, but less pronounced than UiO-66 when the template concentration was below 0.5. mu.g/L. The analysis results are shown in FIG. 4 (b).
As can be seen from examples 4 and 5, the optimization of the metal-organic framework materials for PCR is comprehensive, and not only the specificity but also the sensitivity and yield can be optimized. As can be seen from FIG. 5, ZIF-8 and UiO-66 can adsorb fluorescent single-stranded nucleic acid fragments, resulting in fluorescence quenching; however, the fluorescence is recovered after the complementary fragment is added, and the selective adsorption effect of the complementary fragment and the single-stranded nucleic acid is proved to be an important reason that the metal organic framework material has an improvement effect on the PCR reaction. The metal organic framework material is rich in a pi system from a ligand and positive charges from metal ions. Theoretically, the existence of the pi system enables the pi system to interact with an aromatic group of single-stranded nucleic acid through pi-pi accumulation, and the electropositivity of metal ions enables the metal ions to be mutually attracted with electronegative nucleic acid chains through electrostatic interaction, so that the single strands are adsorbed on the surface of the material, and fluorescence quenching is caused. When the complementary single strand exists, the acting force for forming the double strand is larger than pi-pi accumulation acting force and electrostatic acting force, so that the single strand nucleic acid is dissociated from the metal organic framework material, and the fluorescence is recovered. The selective adsorption of the metal organic framework material to the single-stranded nucleic acid increases the contact area and efficiency between nucleic acid molecules, and is the main reason of the optimization effect of the PCR. The invention can also be used for low copy DNA fragment amplification, long fragment DNA amplification, high GC content DNA fragment amplification or amplification of DNA fragments containing more repetitive sequences.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. The application of the metal organic framework material in polymerase chain reaction is characterized in that the metal organic framework material takes zirconium ions as a metal center and takes terephthalic acid as an organic ligand; or the metal organic framework material takes zinc ions as a metal center and takes 2-methylimidazole as an organic ligand.
2. The use of claim 1, wherein the use is nucleic acid amplification by adding a suspension of the metal-organic framework material to a polymerase chain reaction system.
3. The use of claim 1, wherein the ratio of the mass of the metal organic framework material to the volume of the polymerase chain reaction system is from 10mg/L to 100 mg/L.
4. Use according to claim 1, wherein the template DNA amplified by the polymerase chain reaction is bacteriophage lambda DNA, plasmid DNA, mRNA, animal genomic DNA, plant genomic DNA or human genomic DNA.
5. The use according to claim 4, wherein the lambda phage DNA template concentration is 0.13 μ g/L or more.
6. The use of claim 1, wherein the polymerase chain reaction is a conventional polymerase chain reaction, a reverse transcription polymerase chain reaction, a nested polymerase chain reaction, or a multiplex polymerase chain reaction.
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