CN109423510B - Method for detecting RCA product and application thereof - Google Patents
Method for detecting RCA product and application thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting RCA products and application thereof, in particular to a method for detecting rolling circle amplification products and a method for screening DNA polymerase, wherein the method comprises the following steps: (1) using circular DNA molecules as templates, and performing rolling circle amplification to obtain RCA products; (2) and detecting the RCA product by adopting a particle size analyzer to obtain a particle size analysis result. The method can be directly used for analyzing the grain size of the RCA product, has the advantages of rapidness, accuracy, simple operation and the like, can be used for optimizing components in an RCA system, thereby improving the uniformity of the RCA product, and can also be used for screening DNA polymerase.
Description
Technical Field
The invention relates to the technical field of molecular biology, relates to a method for detecting RCA products and application thereof, and particularly relates to a method for detecting rolling circle amplification products and a method for screening DNA polymerase.
Background
Rolling Circle Amplification (RCA) or multiple strand displacement amplification (MDA) has the characteristics of rapidness, accuracy, less template quantity and the like, and is an important research means in the fields of biomedical technology and biological nanotechnology at present. Phi29 is a DNA polymerase with strand displacement ability, and has been widely used in RCA and MDA. However, RCA or MDA does not control the copy number of the amplification product by controlling the number of cycles, as does the PCR reaction. This is mainly because: (1) the templates have different characteristics, such as different base contents, poly structures and the like, which result in different amplification efficiencies of phi29 for different templates in amplification; (2) the phi29 enzyme is a less stable enzyme, and its activity gradually decreases with the time of reaction, so it is difficult to evaluate more accurately, the copy number of RCA/MDA product in a certain period of time. Based on the above two points, products of RCA and MDA are often not uniform enough, which seriously affects the application of the two technologies.
Existing methods for detecting RCA or MDA products are mainly by electrophoresis, such as agarose electrophoresis and pulse electrophoresis. However, the RCA product generally has a large molecular weight, and when agarose electrophoresis is applied, the macromolecular product is easy to accumulate at the sample application hole, which results in electrophoresis failure. Although the application of pulse electrophoresis solves the defect of the product retention spot sample hole, the application has the disadvantages of complex operation and long time consumption. In addition, the electrophoresis technology has a relatively coarse quantification of the size of the RCA product, and has little significance for accurately researching the uniformity of the RCA product. Moreover, there is also a method of detecting the copy number of the RCA product by a fluorescent quantitative PCR technique, which can perform quantification, but the method is based on the determination of the template or the detection of the RCA product as a whole, and has no guiding significance for the amplification of complex templates.
The RCA product is a multiple-copy single-stranded DNA sequence that can form a "spherical" like structure due to the interaction forces between bases of internal DNA sequences. Fragmented genomic DNA is added with linker sequences and circularized to form single-stranded circular DNA, which can then be amplified by multiple orders of magnitude using rolling circle amplification techniques, resulting in an amplification product known as a DNA Nanoball (DNB). It is known that DNB is a typical RCA product in nature and has the characteristics of an RCA product. Because of differences in the base sequence of the template DNA, differences in the activity of phi29 polymerase and differences in the reaction conditions, RCA products often exhibit different sizes and even morphological features. Because the RCA product is a single-stranded DNA sequence with huge molecular weight, the traditional detection methods, such as gel electrophoresis and a qubit quantitative analyzer, are difficult to distinguish the sizes of different RCA products and the size distribution of the products in a reaction system. The nanometer particle size analyzer adopts a dynamic scattering technology, and quantifies the size of the nanometer particles according to different Brownian motion states of the nanometer particles with different particle sizes by depending on the existing nanometer particle size detection equipment and matched fluorescence signal processing software. However, there is no report on whether RCA products that are not characteristic of nanoparticles can be detected and distinguished by using a particle size analyzer.
Therefore, it is necessary to develop a method for rapidly and accurately detecting the RCA product, and to optimize the RCA reaction system conveniently and efficiently.
Disclosure of Invention
Aiming at the problems of limited means for detecting the RCA product, complex operation, low accuracy and long time consumption of the detection method at present, the invention provides the method for detecting the RCA product and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of detecting an RCA product, comprising the steps of:
(1) using circular DNA molecules as templates, and performing rolling circle amplification to obtain RCA products;
(2) and detecting the RCA product by adopting a particle size analyzer to obtain a particle size analysis result.
In the invention, a particle size analyzer is innovatively used for detecting the RCA products, and the inventor finds that the RCA products with different molecular weights can be distinguished through the detection of the particle size analyzer, and the RCA products with different molecular weights can be accurately distinguished through the obtained particle size distribution diagram, so that the uniformity of the RCA products is improved, and the preparation system of the RCA products is optimized.
According to the invention, RCA products with different molecular weights can be generated by different time of the rolling circle amplification of the circular DNA molecules in the step (1), and the inventor finds that RCA products with different molecular weights can be simultaneously detected by the method, and the time of the rolling circle amplification of the circular DNA molecules is 5-60min, such as 5min, 10min, 20min, 30min, 40min, 50min or 60 min.
It should be noted that the time for rolling circle amplification of circular DNA molecules depends mainly on the DNA polymerase used in the reaction. The reaction time required for rolling circle amplification varies depending on the polymerase and the reaction speed. That is, the reaction time required for the rolling circle amplification may be different between the wild-type DNA polymerase and the mutant DNA polymerase or between different mutant DNA polymerases, and the time for the rolling circle amplification reaction may be adjusted as necessary by those skilled in the art.
According to the invention, after the rolling circle amplification in the step (1), a step of adding a fluorescent molecule modified probe to hybridize with the RCA product is further included.
Preferably, the fluorescent molecule is any one or a combination of at least two of ROX, FAM, HEX, VIC, Cy5 or Cy3, preferably ROX.
In the invention, the nucleotide sequence of the probe modified by the fluorescent molecule for detecting the RCA product is shown as SEQ ID NO.1, and the nucleotide sequence shown as SEQ ID NO.1 is as follows: 5 '-ROXGCTCACAGAAACGACATGGCTACGTCGACTT-3'.
According to the invention, the amount of the fluorescent molecule-modified probe added is 0.5-5. mu.M, and may be, for example, 0.5. mu.M, 0.6. mu.M, 0.7. mu.M, 0.8. mu.M, 0.9. mu.M, 1. mu.M, 1.1. mu.M, 1.2. mu.M, 1.3. mu.M, 1.5. mu.M, 1.8. mu.M, 2. mu.M, 2.3. mu.M, 2.5. mu.M, 2.8. mu.M, 3. mu.M, 3.2. mu.M, 3.5. mu.M, 3.8. mu.M, 4. mu.M, 4.2. mu.M, 4.5. mu.M, 4.8. mu.M or 5. mu.M, preferably 1. mu.M.
According to the invention, the RCA product is a DNA Nanosphere (DNB), the DNB is a DNA Nanosphere (DNB), which is an amplification product produced by amplifying a single-stranded circular DNA by multiple orders of magnitude using RCA technology. The invention adopts different reaction time control to prepare DNB with different molecular weight sizes.
According to the present invention, the particle size analysis result includes any one of or a combination of at least two of a molecular size of the RCA product, a molecular distribution of the RCA product, or a uniformity of the RCA product.
In a specific embodiment, step (1) specifically includes: mu.L of the circularized DNA library was taken out into a PCR tube, and 20. mu.L of DNB preparation buffer was added, and the amplification primer sequences used in the buffer were complementarily paired with the sequences on the circularized DNA library. Shaking and mixing by a vortex oscillator, centrifuging by a mini centrifuge, and placing in a PCR instrument for reaction, wherein the reaction conditions are as follows: 95 deg.C for 1min, 65 deg.C for 1min, 401 min, and 4 deg.C; taking out the PCR tube when the temperature reaches 4 ℃, centrifuging by a mini centrifuge, adding 40 mu L of DNB polymerase mixed solution and 4 mu L of DNB polymerase mixed solution II, shaking and uniformly mixing by a vortex oscillator, centrifuging by the mini centrifuge, immediately placing in a PCR instrument for starting reaction, and reacting under the following conditions: keeping at 30 deg.C for 20min and 4 deg.C; after the reaction, 20. mu.L of termination buffer was added to obtain the RCA product.
According to the present invention, before step (1), a step of preparing a circular DNA molecule is further included, and the preparation method of the circular DNA molecule is a conventional method in the art, and a person skilled in the art can select the circular DNA molecule according to needs, and is not particularly limited herein.
According to the invention, before the step (2), a step of diluting the RCA product obtained in the step (1) is further included.
According to the invention, the dilution is carried out with a diluent which is PBS and/or a loading buffer, preferably a combination of PBS and loading buffer.
According to the present invention, the loading buffer is a nonionic surfactant, preferably a polyoxyethylene polyoxypropylene block copolymer, and more preferably poloxamer 188.
According to the invention, the dilution comprises in particular: mixing the RCA product with a PBS buffer solution to obtain a mixed solution, and adding the loading buffer solution into the mixed solution.
According to the invention, the volume ratio of the RCA product to the PBS buffer is 1: 9-9999, such as 1:9, 1:10, 1:12, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:99, 1:100, 1:120, 1:150, 1:180, 1:200, 1:250, 1:300, 1:350, 1:400, 450, 1:480, 1:499, 1:500, 1:600, 1:700, 1:800, 1:900, 1:950, 1:999, 1:1000, 1:1100, 1:1200, 1:1500, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5500, 1:6000, 1:6500, 1:999, 1: 851: 999, 1:8500, 8000: 3000, 1: 9900, 8000: 1: 990, preferably 1:9999, 1: 9900, 1:8500, 1:1, 1: 9900, 1:99 or preferably 1:99, further preferably 1: 99.
According to the present invention, the volume ratio of the mixed solution to the loading buffer is (100: 10000):1, and may be, for example, 100:1, 110:1, 120:1, 150:1, 180:1, 200:1, 230:1, 250:1, 280:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 1100:1, 1200:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500:1, 4000:1, 4500:1, 5000:1, 5500:1, 6000:1, 6500:1, 7000:1, 7500:1, 8000:1, 8500:1, 9000:1, 90000: 1 or 10000:1, preferably (100: 10000): 1), and more preferably (100: 1), and further preferably (100: 1).
In the invention, the DNB loading buffer solution is added, has a refractive index different from that of an RCA product, ensures that the RCA product of a sample to be detected can be uniformly dispersed in a liquid medium and randomly moves, and obviously improves the detection accuracy.
As a preferred technical solution, the method for detecting an RCA product includes the steps of:
(1) preparing a circular DNA molecule;
(2) using circular DNA molecules as templates, performing rolling circle amplification for 5-60min to obtain RCA products, adding fluorescent molecule modified probes 0.5-5 μ M, and hybridizing with the RCA products to obtain hybrid products;
(3) mixing the hybridization product with PBS buffer solution according to the volume ratio of 1 (9-999) to obtain mixed solution, and adding loading buffer solution with the volume ratio of (100-;
(4) and detecting the hybridization product by adopting a particle size analyzer to obtain a particle size analysis result.
In another aspect, the present invention provides a method for screening a DNA polymerase, comprising the steps of:
(1') performing rolling circle amplification by using different DNA polymerases by using the circular DNA molecules as templates to obtain RCA products;
(2') detecting the RCA product by adopting a particle size analyzer to obtain a particle size analysis result, and then screening a proper DNA polymerase;
wherein the DNA polymerase has strand displacement activity.
In the invention, through the analysis of DNBs with different sizes, the DNA polymerases aiming at the same template can be accurately screened out to polymerize the DNBs with different sizes under the same reaction condition, and then the proper DNA polymerase is selected according to the DNB size required by the experiment purpose.
According to the present invention, the different DNA polymerases of step (1') have different amplification efficiencies due to their different binding to the template, thereby producing DNBs of different sizes, and those skilled in the art can produce DNBs according to their needs without any particular limitation.
According to the present invention, the DNA polymerase in step (1') may be any DNA polymerase capable of performing RCA, and the more DNA polymerases selected in the present invention is phi29DNA polymerase including wild-type DNA polymerase and/or mutant DNA polymerase, the larger the range of DNA polymerase to be screened, and the larger the range of DNA polymerase to be screened, the skilled person can select the DNA polymerase as needed.
In a specific embodiment, step (1') specifically comprises: taking 20 mu L of cyclized DNA library to a plurality of PCR tubes, adding 20 mu L of DNB to prepare buffer solution, shaking and uniformly mixing the buffer solution by a vortex oscillator, centrifuging the mixture by a centrifuge, and placing the mixture in a PCR instrument for reaction under the following reaction conditions: 95 deg.C for 1min, 65 deg.C for 1min, 40 deg.C for 1min, and 4 deg.C; taking out the PCR tubes when the temperature reaches 4 ℃, centrifuging by a centrifuge, adding 40 mu of LDNA polymerase reaction mixed liquor and 4 mu of different mutant phi29 polymerases into each reaction tube, shaking and uniformly mixing by a vortex oscillator, centrifuging by the centrifuge, immediately placing in a PCR instrument for starting reaction, and reacting under the following conditions: keeping at 30 deg.C for 20min and 4 deg.C; after the reaction, 20. mu.L of termination buffer was added to obtain the RCA product.
According to the invention, the time of rolling circle amplification in step (1') is 5-60min, for example, 5min, 10min, 20min, 30min, 40min, 50min or 60min, and the DNA polymerase is screened by different RCA products by performing rolling circle amplification for the same time by using different DNA polymerases.
According to the invention, after the rolling circle amplification in step (1'), further comprising a step of adding a fluorescent molecule-modified probe to hybridize with the RCA product;
preferably, the fluorescent molecule is any one or a combination of at least two of ROX, FAM, HEX, VIC, Cy5 or Cy3, preferably ROX.
According to the invention, the fluorescent molecule-modified probe is added in an amount of 0.5-5. mu.M, for example, 0.5. mu.M, 0.6. mu.M, 0.7. mu.M, 0.8. mu.M, 0.9. mu.M, 1. mu.M, 1.1. mu.M, 1.2. mu.M, 1.3. mu.M, 1.5. mu.M, 1.8. mu.M, 2. mu.M, 2.3. mu.M, 2.5. mu.M, 2.8. mu.M, 3. mu.M, 3.2. mu.M, 3.5. mu.M, 3.8. mu.M, 4. mu.M, 4.2. mu.M, 4.5. mu.M, 4.8. mu.M or 5. mu.M, preferably 1. mu.M.
According to the invention, the RCA product is a DNA Nanosphere (DNB), which is an amplification product generated by amplifying a single-stranded circular DNA by multiple orders of magnitude using RCA technology. The difference in the reaction time of rolling circle amplification results in the production of DNB of different molecular weight sizes.
According to the present invention, the particle size analysis result includes any one of a molecular size of the RCA product, a molecular distribution of the RCA product, or a uniformity of the RCA product, or a combination of at least two of them.
According to the present invention, the preparation method of the circular DNA library in step (1') is a conventional method in the art, and a person skilled in the art can select the circular DNA library as required, and is not particularly limited herein.
According to the invention, before the step (2), the method further comprises a step of diluting the RCA product obtained in the step (1).
According to the invention, the dilution is carried out with a diluent which is PBS and/or a loading buffer, preferably a combination of PBS and loading buffer.
According to the present invention, the loading buffer is a nonionic surfactant, preferably a polyoxyethylene polyoxypropylene block copolymer, and more preferably poloxamer 188.
According to the invention, said dilution comprises in particular: mixing the RCA product with a PBS buffer solution to obtain a mixed solution, and adding the loading buffer solution into the mixed solution.
According to the invention, the volume ratio of the RCA product to the PBS buffer is 1: 9-9999, such as 1:9, 1:10, 1:12, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:99, 1:100, 1:120, 1:150, 1:180, 1:200, 1:250, 1:300, 1:350, 1:400, 450, 1:480, 1:499, 1:500, 1:600, 1:700, 1:800, 1:900, 1:950, 1:999, 1:1000, 1:1100, 1:1200, 1:1500, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1: 995500, 1:6000, 1:6500, 1:999, 1: 851: 2000, 1:8000, 1:9500, preferably 1:3000, 1:4000, 1:4500, 1: 9955000, 1: 9900, 1:8000, 1:9500, 1:99 or preferably 1:3000, further preferably 1: 99.
According to the present invention, the volume ratio of the mixed solution to the loading buffer is (100: 10000):1, and may be, for example, 100:1, 110:1, 120:1, 150:1, 180:1, 200:1, 230:1, 250:1, 280:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 1100:1, 1200:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500:1, 4000:1, 4500:1, 5000:1, 5500:1, 6000:1, 6500:1, 7000:1, 7500:1, 8000:1, 8500:1, 9000:1, 90000: 1 or 10000:1, preferably (100: 10000): 1), and more preferably (100: 1), and further preferably (100: 1).
In the invention, the DNB loading buffer solution with the refractive index different from that of the RCA product is added, so that the RCA product of a sample to be detected can be uniformly dispersed in a liquid medium and randomly moves, and the detection accuracy is obviously improved.
As a preferred technical scheme, the screening method of phi29 polymerase comprises the following steps:
(1') preparing a circular DNA molecule;
(2') performing rolling circle amplification for 5-60min by using different DNA polymerases by using a circular DNA molecule as a template to obtain an RCA product, adding a probe modified by a fluorescent molecule of 0.5-5 mu M, and hybridizing with the RCA product to obtain a hybridized product;
(3') mixing the hybridization product with a PBS buffer solution according to a volume ratio of 1 (99-999) to obtain a mixed solution, and adding a loading buffer solution with the volume ratio of (100-;
(4') detecting the hybridization product by using a particle size analyzer to obtain a particle size analysis result, and then screening a proper DNA polymerase.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the method can be directly used for analyzing the grain diameter of the RCA product, has the advantages of rapidness, accuracy, simple and convenient operation and the like, and can be used for optimizing components in an RCA system, thereby improving the uniformity of the RCA product;
(2) the method can be used for high-throughput screening of DNA polymerase mutants suitable for different DNA templates, and can improve the synthesis efficiency by combining a second-generation sequencing means.
Drawings
FIG. 1 shows the results of particle size distributions of DNB prepared from mutant phi29 polymerase and wild-type phi29 polymerase, where Median line is Median, Mean is Mean, and Outliers is outlier;
FIG. 2(A) the signal distribution results of DNB prepared by wild-type phi29 polymerase, and FIG. 2(B) the signal distribution results of DNB prepared by mutant phi29 polymerase;
FIG. 3 is a graph showing the results of Q30, efficiency of transfection and effective DNB ratio of DNB prepared by mutant phi29 polymerase and DNB prepared by wild-type phi29 polymerase.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Reagents used in the following examples:
1. SE50 kit: a library building kit and a single end-on-machine sequencing kit which are matched with a sequencing instrument BGI-SEQ500RS from Huada gene.
2. 10% poloxamer 188: under the trade name pluronic F68, available from sigma under the trade name P5556.
3. 1 XPBS: purchased from Thermo Fisher, ph 7.4.
Example 1: RCA product detection method
(1) Preparation of circularized template DNA sequence: the preparation of circularized template DNA was performed according to the library construction kit of SE50 kit of the BGI-SEQ500RS platform and the methods provided in the kit instructions. The method comprises the following specific steps: extracting the genome of the Escherichia coli by using a kit, breaking the genome DNA by a physical breaking mode, running a DNA gel, and recovering a DNA fragment with the fragment size of 150-250bp to prepare a mixed library. Taking 160ng of DNA mixed library, supplementing the volume to 48 mu L with molecular water, fully mixing uniformly, centrifuging for a short time, placing on a PCR instrument for incubation for 5min at 95 ℃, taking out a PCR tube immediately after incubation, placing on ice for cooling, adding 11.6 mu L of 1 mu M cyclization sequence (from an SE50 kit) and 0.5 mu L of ligase into the PCR tube, fully mixing uniformly, centrifuging for a short time, and incubating for 30min at 37 ℃;
(2) preparation of DNB at different reaction times: respectively taking 20 mu L of the cyclized DNA library to three 0.2mL PCR tubes, respectively adding 20 mu L of DNB preparation buffer solution (derived from DNB preparation kit in SE50 kit), shaking and uniformly mixing by using a vortex oscillator, centrifuging by using a mini centrifuge for 5s, and placing the mixture in a PCR instrument for reaction under the reaction conditions that: 95 deg.C for 1min, 65 deg.C for 1min, 40 deg.C for 1min, and 4 deg.C; taking out the PCR tube when the temperature reaches 4 ℃, centrifuging the PCR tube for 5s by using a mini centrifuge, adding 40 mu L of DNB polymerase reaction solution I and 4 mu L of DNNB polymerase II (from DNB preparation kit in SE50 kit), shaking and uniformly mixing the mixture by using a vortex oscillator, centrifuging the mixture for 5s by using the mini centrifuge, placing the mixture in a PCR instrument for starting reaction under the reaction conditions: at 30 deg.C for 5min/20min/30min (total 3 groups: group A reacting for 5min, group B reacting for 20min, and group C reacting for 30min), and maintaining at 4 deg.C; after the DNB reaction is finished in 5min, 20 mu L of termination buffer solution (0.5M EDTA) is added and mixed gently; after 20min of DNB reaction, 20. mu.L of termination buffer (0.5M EDTA) was added in the same manner, and the reaction was terminated by gently mixing; after the DNB reaction is finished for 30min, 20 mu L of termination buffer solution (0.5M EDTA) is added in the same way, and the reaction is terminated by gently mixing the solution uniformly; in order to ensure the accurate detection of a sample to be detected, the sample is diluted by PBS and 10% poloxamer 188; taking 100 μ L of 5min DNB (group A), 20min DNB (group B) and 30min DNB (group C), respectively adding 9900 μ L of PBS and 100 μ L of 10% poloxamer 188, and gently mixing; adding 1 μ M probe with fluorescent modification (probe sequence: 5 '-ROX GCTCACAGAACGACATGGCATCGATCCGACTT-3' (SEQ ID NO.1)) into the prepared three DNB solutions, carrying out complementary pairing hybridization with DNB, incubating at room temperature for 30min, and repeating each group of reactions twice to verify repeatability;
(3) detecting by a nano particle size analyzer: turning on a particle size analyzer (Malvern NanoSight NS300), preheating for 30min, sequentially adding the hybridized 5min DNB, 20min DNB and 30min DNB mixed solution into a detector for detection, and performing data analysis on the peak intensity, peak area and particle size distribution range in the obtained particle size distribution diagram, wherein the results are shown in the following table 1.
TABLE 1
D10-10% of the particles in the sample have a diameter less than this value
D50-the 50% particle diameter in the sample is less than this value
D90-the 90% particle diameter in the sample is less than this value
As can be seen from the DNB average particle size at different times in table 1, the 30min DNB is greater than 20min DNB is greater than 5min DNB, indicating that this particle size analyzer is capable of accurately distinguishing DNBs of different sizes. As can be seen from the particle size distributions D10, D50 and D90 of the different samples, the increase rate of the particle size of DNB after 20min of rolling circle replication was significantly smaller than that of the first 20min, indicating that the amplification efficiency of phi29DNA polymerase after 20min had begun to decrease. In addition, as the RCA time is longer, the number of particles is larger in DNB concentration, but as the standard deviation of particle size is found, the longer the RCA time is, the more uneven the particle size distribution is. The information is beneficial to guiding the system optimization of the subsequent DNB preparation, preparing more uniform DNB and further improving the sequencing quality.
Example 2 screening of phi29 polymerase
(1) Preparing a mixed library according to step (1) of reference example 1;
(2) different DNB solutions were prepared using different phi29 polymerases: taking 20 mu L of the cyclized DNA library to two 0.2mL PCR tubes respectively, adding 20 mu L of DNB preparation buffer (from DNB preparation kit in SE50 kit) into each PCR tube respectively, shaking and mixing uniformly by a vortex oscillator, centrifuging by a mini centrifuge for 5s, and placing in a PCR instrument for reaction under the following reaction conditions: 1min at 95 ℃, 1min at 65 ℃, 1min at 40 ℃ and keeping at 4 ℃; taking out the PCR tube when the temperature reaches 4 ℃, centrifuging the PCR tube for 5s by using a mini centrifuge, adding 40 mu L of DNA polymerase reaction solution I (from a DNB preparation kit in an SE50 kit) and 4 mu L of different phi29 polymerases (namely wild phi29 polymerase and mutant phi29 polymerase to be screened), shaking and uniformly mixing the polymerases in a vortex oscillator, centrifuging the PCR tube for 5s by using the mini centrifuge, immediately placing the mixture in a PCR instrument for starting reaction, wherein the reaction conditions are as follows: keeping at 30 deg.C for 20min and 4 deg.C; after 20min of DNB reaction, 20 μ L of termination buffer (0.5M EDTA) was added and mixed gently for further use;
(3) adding 9900 mu L of PBS and 100 mu L of 10% poloxamer 188 into DNB after reaction, and diluting DNB solutions generated by different polymerase reactions to proper concentrations; then, 1. mu.M of a probe (probe sequence: 5 '-ROX GCTCACAGAACGACATGGCTACGATCCGACT-3' (SEQ ID NO.1)) with fluorescent modification was added, and complementary pair hybridization was performed with DNB, followed by incubation at room temperature for 30 min;
(4) taking 2 mu L of the DNB solution after hybridization respectively to carry out Qubit quantification, wherein the concentration of the DNB solution in which the wild type phi29 polymerase is positioned is 27.3 ng/mu L, and the concentration of the DNB solution in which the mutant type phi29 polymerase to be screened is 25 ng/mu L;
(5) detecting by a nanometer particle size analyzer, detecting DNB by the particle size analyzer, and distinguishing DNB with different sizes by the obtained particle size distribution diagram so as to screen proper phi29 polymerase, wherein the result is shown in figure 1; (6) and (3) respectively loading the DNBs in the step (2) on the chips in the same batch according to the DNB loading method of BGISEQ500, carrying out 1-cycle sequencing detection on the BGI-seq500, and obtaining the different signal distributions and the loading conditions of the DNBs on the chips by the data analysis software of the BGI-seq500, wherein the results are shown in FIGS. 2(A) -2 (B) and 3.
As can be seen from fig. 1, the DNB profiles prepared with mutant polymerases both showed more concentration than that of the wild type from the DNB profiles of 10% to 90% and 1% to 99%; furthermore, it can be seen from the outliers, both the outliers and median numbers of DNBs prepared using the mutant polymerases were lower than those of the wild type. Overall, the mutant polymerases produced more uniform DNBs.
As can be seen from fig. 2(a) -2 (B), the DNB signals produced by the mutant phi29 polymerase were more evenly distributed, while the DNBs produced by the wild-type phi29 polymerase were more diffuse.
As can be seen from fig. 3, DNBs prepared by mutant phi29 polymerase have higher loading efficiency than DNBs prepared by wild-type phi29 polymerase, and among the DNBs loaded on the chip, the DNBs prepared by mutant phi29 polymerase have a larger effective ratio for subsequent sequencing, and the quality value (Q30) is higher than that of the wild-type DNBs, which means that the mutant DNBs can exhibit better sequencing quality.
From the above results, it is demonstrated that the correspondence between the traditional qubit quantitative instrument and the results of the on-computer test is not large, even if the test results of the qubit quantitative instrument show high-concentration DNB, no higher signal value is shown on the sequencer, and the results of the nanoparticle size analyzer and the data results on the sequencer show that DNB prepared by the mutant is more uniform, and better sequencing quality can be obtained, which indicates that the nanoparticle size analyzer can rapidly and effectively screen out proper phi29 polymerase.
Comparative example DNB loading buffered 10% poloxamer 188 was used
(1) Preparation of circularized template DNA sequence: the concrete preparation steps are the same as the step (1) of example 1 to prepare a mixed library;
(2) preparation of DNB: taking 20 mu L of the cyclized DNA library to two 0.2mL PCR tubes respectively, adding 20 mu L of DNB preparation buffer (from DNB preparation kit in SE50 kit) into each PCR tube respectively, shaking and mixing uniformly by a vortex oscillator, centrifuging by a mini centrifuge for 5s, and placing in a PCR instrument for reaction under the following reaction conditions: 1min at 95 ℃, 1min at 65 ℃, 1min at 40 ℃ and keeping at 4 ℃; when the temperature reaches 4 ℃, taking out the PCR tube, centrifuging for 5s by a mini-centrifuge, adding 40 mu L of DNA polymerase reaction solution I (from a DNB preparation kit in an SE50 kit) and 4 mu L of polymerase 29, shaking and uniformly mixing by a vortex oscillator, centrifuging for 5s by the mini-centrifuge, placing in a PCR instrument for starting reaction, and reacting under the following conditions: keeping at 30 deg.C for 20min and 4 deg.C; after the 20min DNB reaction was complete, 20. mu.L of stop buffer (0.5M EDTA) was added and gently mixed. Taking 50 mu L of DNB for 20min, adding 5000 mu L of PBS, and lightly mixing uniformly to mark as a sample D; 4950 μ L PBS and 50 μ L10% poloxamer 188 were added to the remaining 50 μ L DNB, mixed gently, labeled as sample E, and two replicates were performed on sample D and sample E to verify reproducibility;
(3) detecting by a nano particle size analyzer: turning on a particle size analyzer (Malvern NanoSight NS300), preheating for 30min, sequentially adding sample D and sample E into the detector for detection, and analyzing the peak intensity, peak area and particle size distribution range of the obtained particle size distribution diagram, with the results shown in Table 2.
TABLE 2
D10: 10% of the particles in the sample having a diameter smaller than this value
D50: 50% of the particles in the sample have a diameter smaller than this value
D90: 90% of the particles in the sample have a diameter smaller than this value
As can be seen from the results of the examples and comparative examples in Table 2, samples D1 and D2 had poor reproducibility of the two samples due to the absence of the addition of the loading buffer of the present invention, and the measured particle size difference was large, and the difference in the number of particles per unit volume was also large, whereas samples E1 and E2 to which the loading buffer of the present invention was added had good reproducibility of data.
In conclusion, the method can be directly used for analyzing the grain size of the RCA product, has the advantages of rapidness, accuracy, simplicity and convenience in operation and the like, and can be used for optimizing components in an RCA system, so that the uniformity of the RCA product is improved; moreover, the method can also be used for high-throughput screening of DNA polymerase mutants suitable for different DNA templates, and can remarkably improve the synthesis efficiency by combining a second-generation sequencing means.
The applicant states that the present invention is illustrated by the above examples to show the details of the process equipment and process flow of the present invention, but the present invention is not limited to the above details of the process equipment and process flow, which means that the present invention must not be implemented by relying on the above details of the process equipment and process flow. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (44)
1. A method of detecting an RCA product, comprising the steps of:
(1) using circular DNA molecules as templates, and performing rolling circle amplification to obtain RCA products;
diluting the RCA product with a diluent comprising a non-ionic surfactant;
the nonionic surfactant is poloxamer 188;
(2) and detecting the RCA product by adopting a particle size analyzer based on a dynamic scattering technology to obtain a particle size analysis result.
2. The method of claim 1, wherein the RCA products of step (1) have different molecular weight sizes.
3. The method of claim 2, wherein RCA products with different molecular weights are prepared by controlling different rolling circle amplification times.
4. The method of claim 1, wherein the rolling circle amplification time is 5-60 min.
5. The method of claim 4, wherein the rolling circle amplification time is 5-30 min.
6. The method of claim 5, wherein the rolling circle amplification is performed for a period of 5-20 min.
7. The method of claim 1, further comprising, after said rolling circle amplification of step (1), the step of adding a fluorescent molecule-modified probe to hybridize to said RCA product.
8. The method of claim 7, wherein the fluorescent molecule is any one of or a combination of at least two of ROX, FAM, HEX, VIC, Cy5, or Cy 3.
9. The method of claim 8, wherein the fluorescent molecule is ROX.
10. The method of claim 7, wherein the fluorescent molecule-modified probe is added in an amount of 0.5-5 μ M.
11. The method of claim 10, wherein the fluorescent molecule-modified probe is added in an amount of 1 μ M.
12. The method of claim 1, wherein said RCA product is a DNA nanosphere.
13. The method of claim 1, wherein the particle size analysis results comprise any one of, or a combination of at least two of, the molecular size of the RCA products, the molecular distribution of the RCA products, or the homogeneity of the RCA products.
14. The method of claim 1, further comprising the step of preparing a circular DNA molecule prior to step (1).
15. The method of claim 1, wherein the diluent is a combination of PBS and loading buffer.
16. The method according to claim 15, wherein the diluting specifically comprises: mixing the RCA product with a PBS buffer solution to obtain a mixed solution, and adding the loading buffer solution into the mixed solution.
17. The method of claim 16, wherein the volume ratio of the RCA product to the PBS buffer is 1 (9-9999).
18. The method of claim 17, wherein the volume ratio of the RCA product to the PBS buffer is 1 (99-999).
19. The method of claim 18, wherein the volume ratio of the RCA product to the PBS buffer is 1: 99.
20. The method as claimed in claim 18, wherein the volume ratio of the mixed solution to the loading buffer is (100-10000): 1.
21. The method as claimed in claim 20, wherein the volume ratio of the mixed solution to the loading buffer is (100-1000): 1.
22. The method of claim 21, wherein the volume ratio of the mixture to the loading buffer is 100: 1.
23. A method for screening a DNA polymerase, comprising the steps of:
(1') adopting circular DNA molecules as templates and adopting different DNA polymerases to carry out rolling circle amplification to obtain an RCA product;
diluting the RCA product with a diluent comprising a non-ionic surfactant;
the nonionic surfactant is poloxamer 188;
(2') detecting the RCA product by adopting a particle size analyzer based on a dynamic scattering technology to obtain a particle size analysis result, and then screening appropriate DNA polymerase;
wherein the DNA polymerase has strand displacement activity.
24. The method of claim 23, wherein the DNA polymerase of step (1') comprises a wild-type DNA polymerase and/or a mutant DNA polymerase.
25. The method of claim 24, wherein the DNA polymerase is phi29DNA polymerase.
26. The method of claim 23, wherein the rolling circle amplification time is 5-60 min.
27. The method of claim 26, wherein the rolling circle amplification time is 5-30 min.
28. The method of claim 27, wherein the rolling circle amplification time is 5-20 min.
29. The method of claim 23, further comprising, after said rolling circle amplification of step (1'), the step of adding a fluorescent molecular modification probe to hybridize to said RCA product.
30. The method of claim 29, wherein the fluorescent molecule is any one of or a combination of at least two of ROX, FAM, HEX, VIC, Cy5, or Cy 3.
31. The method of claim 30, wherein the fluorescent molecule is ROX.
32. The method of claim 29, wherein the fluorescent molecule-modified probe is added in an amount of 0.5-5 μ M.
33. The method of claim 32, wherein the fluorescent molecule-modified probe is added in an amount of 1 μ M.
34. The method of claim 23, wherein said RCA product is a DNA nanosphere.
35. The method of claim 23, wherein the particle size analysis results comprise any one of, or a combination of at least two of, the molecular size of the RCA products, the molecular distribution of the RCA products, or the homogeneity of the RCA products.
36. The method of claim 23, further comprising the step of preparing a circular DNA molecule prior to step (1').
37. The method of claim 23, wherein the diluent is a combination of PBS and loading buffer.
38. The method according to claim 37, wherein the diluting comprises: mixing the RCA product with a PBS buffer solution to obtain a mixed solution, and adding the loading buffer solution into the mixed solution.
39. The method of claim 38, wherein the volume ratio of the RCA product to the PBS buffer is 1 (9-9999).
40. The method of claim 39, wherein the volume ratio of the RCA product to the PBS buffer is 1 (99-999).
41. The method of claim 40, wherein the volume ratio of the RCA product to the PBS buffer is 1: 99.
42. The method as claimed in claim 38, wherein the volume ratio of the mixed solution to the loading buffer is (100-10000): 1.
43. The method as claimed in claim 42, wherein the volume ratio of the mixed solution to the loading buffer is (100-1000): 1.
44. The method of claim 43, wherein the volume ratio of the mixture to the loading buffer is 100: 1.
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