CN104845965A - Method for improving amplification efficiency of rolling circle amplification (RCA) by utilizing poly compound - Google Patents
Method for improving amplification efficiency of rolling circle amplification (RCA) by utilizing poly compound Download PDFInfo
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Abstract
The invention provides a method for improving RCA amplification efficiency by utilizing a poly compound. The method comprises the following steps: respectively preparing a solution PEG 3350 with a concentration of 0.1 g/ml, a solution mPEG-SC 2000 with a concentration of 0.2 mg/l and a solution mPEG-SC 5000 with a concentration of 0.1 mg/l; and amplifying a kit by using a TempliPhi DNA sequencing template, carrying out mutation operation and cultivation operation according to instructions on the kit, adding polyethylene glycol or a derivative thereof with a certain concentration of in the process of rolling circle amplification, and respectively subjecting RCA amplification products to electrophoresis in agarose gel with a concentration of 1%. The method for improving RCA amplification efficiency by utilizing the poly compound provided by the invention can significantly improve RCA amplification efficiency, strengthens the sensitivity of RCA and realizes further optimization of a commercial RCA kit.
Description
Technical field
The present invention relates to a kind of method utilizing poly-compounds to improve RCA amplification efficiency.
Background technology
Nucleic acid amplification technologies is a basic modern biotechnology, is widely used in the aspects such as life science, medical treatment, agricultural, environmental monitoring, legal medical expert's evidence obtaining.Its application is usually based on the detection of nucleic acid, qualification, quantitatively and sequential analysis, this just requires that nucleic acid amplification technologies has high susceptibility, specificity and accuracy.Due to the sample DNA that obtains in organism minute quantity often, therefore, develop high efficiency nucleic acid amplification technologies very crucial.
Nineteen eighty-three, Mullis etc. have invented polymerase chain reaction (polymerase chain reaction, PCR) technology, and this technology achieves DNA cloning by the continuous circulation of some primitive reactions, and the output of DNA is constantly increased with exponential form.But PCR method still have weak point as: PCR process need repeatedly thermal cycling cannot break away from the limitation relying on superior plant and instrument; Easy generation non-specific amplification and cause producing false positive or false negative result; Be not suitable for whole genome amplification etc.
Compared with round pcr, many isothermal amplification technologies of at present exploitation have rapidly and efficiently, and avoid the advantage such as to need to thermal cycler.These technology comprise dependence amplification of nucleic acid sequences (nucleic acidsequence based amplification, NASBA), transcript mediated amplification (transcription-mediatedamplification, TMA), ring mediated isothermal amplification (1oop-mediated isothermalamplification, LAMP), chain substitutes amplification (strand displacement amplification, SDA), desmolase amplification (helicase-dependent amplification, and rolling circle amplification (rollingcircle amplification HDA), RCA) etc.Wherein, rolling circle amplification is as the simple nucleic acid amplification means efficiently of one, have that reaction conditions gentleness, design of primers are simple, the prime end advantages such as easily fixing and product analysis means are various, be widely used in nucleic acid sequencing, whole genome amplification, gene diagnosis, single nucleotide polymorphism, ProteinChip Analysis etc. at present.
In recent years, based on the TempliPhi that many primers RCA technology of bacteriophage Phi29DNA polysaccharase and hexabasic base random primer is produced
tMdNA sequencing template amplification test kit, from the parent material of 1-100pgDNA, can obtain the highly purified DNA of 3-4 μ g through reaction in 4-6 hour.This test kit can increase the large cyclic DNA such as plasmid, phage DNA, bacterial artificial chromosome (Bacteria artificialchromosome, BAC), clay, for the circular template that cannot clone, also can carry out in-vitro multiplication by this method.And the high quality DNA product obtained is the tandem-repeated copies of the amplification template added, is the linear double-strand of high molecular, order-checking can be directly used in, enzyme cuts or other clones, mark and the aspect such as detection.
But, in actual applications, the problems such as existing RCA technology still exists certain deficiency, and as slow in speed of response, sensitivity is low.Especially be embodied in the early diagnosis of virus, because DNA profiling in reaction system is complicated or content is very low, a large amount of primer effectively can not match with template, makes easily to form dimeric structure between primer, causes the generation of mispairing, thus reduces the amplification efficiency of RCA.At present, a kind of single strand binding protein TthSSB that uses is had to improve RCA amplification efficiency method.But the method needs first to be purified into activated TthSSB albumen, complex steps, to waste time and energy.Therefore, in the art, a kind of method developing simple, fast and efficient raising RCA amplification efficiency is needed.
Summary of the invention
The technical problem to be solved in the present invention, is to provide a kind of method utilizing poly-compounds to improve RCA amplification efficiency.
The present invention is achieved in that a kind of method utilizing poly-compounds to improve RCA amplification efficiency, comprises the following steps:
(1) configuration concentration is mPEG-SC 5,000 three kinds of solution of the PEG 3350 of 0.1g/ml, the mPEG-SC 2000 of 0.2mg/l and 0.1mg/l respectively, for subsequent use;
(2) TempliPhi is used
tMdNA sequencing template amplification test kit:
A. sex change: using pUC 19 plasmid DNA as template, adds 1ng pUC 19 plasmid DNA in 5 μ l sample buffers, and after mixing, 95 DEG C of heating 3min, are then cooled to room temperature or 4 DEG C, obtain denaturing sample liquid;
B. cultivate: in 5 μ l reaction buffers, add 0.2 μ l Phi29DNA polysaccharase, mixing, is placed on ice, obtains premix synthase liquid; Add in described denaturing sample liquid by 5 μ l premix synthase liquid, mixing, obtains RCA reaction solution; PEG 3350 solution of preset vol, mPEG-SC 2000 solution, mPEG-SC 5000 solution is added respectively in each RCA reaction solution; Each RCA reaction solution is respectively at water-bath 4-18h at 30 DEG C; 65 DEG C of heating 10min, are cooled to 4 DEG C; 3 μ l RCA products are got 3 μ l reaction mixtures with 1% sepharose carry out electrophoresis in 1% sepharoses respectively.
Further, in described step B, PEG 3350 solution add-on is 0.5 μ l, 2 μ l or 4 μ l.
Further, in described step B, mPEG-SC 2000 solution add-on is 0.5 μ l, 1 μ l or 1.5 μ l.
Further, in described step B, mPEG-SC 5000 solution add-on is 0.5 μ l, 1.5 μ l or 3 μ l.
The invention has the advantages that: by adding certain density PEG or derivatives thereof in RCA reaction process, the efficiency of rolling circle amplification can be improved significantly, strengthening the sensitivity of rolling circle amplification, achieving the further optimization to commercially available RCA test kit.
Accompanying drawing explanation
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 be in the present invention with pUC 19 plasmid DNA for template, PEG 3350 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Fig. 2 be in the present invention with pUC 19 plasmid DNA for template, mPEG-SC 2000 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Fig. 3 be in the present invention with pUC 19 plasmid DNA for template, mPEG-SC 5000 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Fig. 4 be in the present invention with the pUC 19 plasmid DNA reaction system that is template add RCA amplified production enzyme that PEG 3350 obtains cut after electrophoresis schematic diagram.
Fig. 5 be in the present invention with the pUC 19 plasmid DNA reaction system that is template add RCA amplified production enzyme that mPEG-SC2000 obtains cut after electrophoresis schematic diagram.
Fig. 6 be in the present invention with the pUC 19 plasmid DNA reaction system that is template add RCA amplified production enzyme that mPEG-SC5000 obtains cut after electrophoresis schematic diagram.
Fig. 7 be in the present invention with mono-clonal (containing recombinant plasmid pUC19-hip) for template, PEG 3350 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Fig. 8 be in the present invention with mono-clonal (containing recombinant plasmid pUC19-hip) for template, mPEG-SC2000 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Fig. 9 be in the present invention with mono-clonal (containing recombinant plasmid pUC19-hip) for template, mPEG-SC5000 affects the electrophoresis schematic diagram of situation to RCA amplification efficiency.
Figure 10 is that the reaction system of template is added the RCA amplified production that PEG 3350 obtains and carried out the electrophoresis schematic diagram after PCR with mono-clonal in the present invention.
Figure 11 is that the reaction system of template is added the RCA amplified production that mPEG-SC 2000 obtains and carried out the electrophoresis schematic diagram after PCR with mono-clonal in the present invention.
Figure 12 is that the reaction system of template is added the RCA amplified production that mPEG-SC 5000 obtains and carried out the electrophoresis schematic diagram after PCR with mono-clonal in the present invention.
Embodiment
Embodiment 1
Utilize poly-compounds to improve a method for RCA amplification efficiency, comprise the following steps:
(1) configuration concentration is mPEG-SC 5000 (mono methoxy polyethylene glycol-succinimdyl carbonate 5000) three kinds of solution of the PEG 3350 (PEG3350) of 0.1g/ml, the mPEG-SC 2000 (mono methoxy polyethylene glycol-succinimdyl carbonate 2000) of 0.2mg/l and 0.1mg/l respectively, for subsequent use;
(2) TempliPhi of Amersham Biosciences company is used
tMdNA sequencing template amplification test kit, operates according to the explanation of test kit:
A. sex change: using pUC 19 plasmid DNA as template, adds 1ng pUC 19 plasmid DNA in 5 μ l sample buffers, and after mixing, 95 DEG C of heating 3min, are then cooled to room temperature or 4 DEG C, obtain denaturing sample liquid;
B. cultivate: in 5 μ l reaction buffers, add 0.2 μ l Phi29DNA polysaccharase, mixing, is placed on ice, obtains premix synthase liquid; Add in described denaturing sample liquid by 5 μ l premix synthase liquid, mixing, obtains RCA reaction solution; PEG 3350 solution of preset vol, mPEG-SC 2000 solution, mPEG-SC 5000 solution is added respectively in each RCA reaction solution; Each RCA reaction solution is respectively at water-bath 4-18h at 30 DEG C; 65 DEG C of heating 10min, are cooled to 4 DEG C; Described PEG 3350 solution add-on is 0.5 μ l, 2 μ l or 4 μ l; Described mPEG-SC 2000 solution add-on is 0.5 μ l, 1 μ l or 1.5 μ l; Described mPEG-SC 5000 solution add-on is 0.5 μ l, 1.5 μ l or 3 μ l.
C.RCA amplified production detects: get 3 μ l RCA products with 1% sepharose for electrophoresis supporting dielectric, electrophoresis 30min under 1 × TAE and 120V voltage, then dye 15min in 0.5 μ g/mL ethidium bromide staining liquid, observe with ultraviolet transilluminator, specific experiment the results are shown in Figure 1, Fig. 2 and Fig. 3.Sequence after pUC 19 plasmid DNA RCA increases is as shown in SEQ ID NO:3.
The electrophoresis result of above-mentioned each RCA amplified production is shown in Fig. 1, Fig. 2 and Fig. 3.Wherein, in Fig. 1, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,2,4 PEG 3350 solution being illustrated respectively in RCA reaction system the 0.1g/ml adding 0.5 μ l, 2 μ l and 4 μ l.In Fig. 2, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,1,1.5 mPEG-SC 2000 solution being illustrated respectively in RCA reaction solution the 0.2mg/l adding 0.5 μ l, 1 μ l and 1.5 μ l.In Fig. 3, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,1.5,3 mPEG-SC 5000 solution being illustrated respectively in this RCA reaction system the 0.1mg/l adding 0.5 μ l, 1.5 μ l and 3 μ l.
As can be seen from Fig. 1, Fig. 2 and Fig. 3, along with the increase of the PEG 3350 added, mPEG-SC 2000 and mPEG-SC 5000 volume, DNA band obviously thicker, brighten, DNA rolling circle amplification efficiency significantly improves, reach a certain amount of after, the amplification of RCA product starts to be suppressed.
Embodiment 2
The each RCA product (rolling circle amplification product of pUC 19 plasmid DNA) embodiment 1 obtained carries out restriction analysis, comprises the following steps:
(1) according to the form below preparation endonuclease reaction liquid 30 μ l:
| Reagent | Usage quantity (μ l) |
| HindIII | 1 |
| 10×Fast Digest Buffer | 2 |
| The RCA product of embodiment 1 | 1.2 |
| Sterile purified water | 25.8 |
| Cumulative volume | 30 |
(2) endonuclease reaction is carried out by following condition: 37 DEG C of 35min; 80 DEG C of 10min;
(3) enzyme cuts after product detection: get 7 μ l endonuclease reaction liquid with 1% sepharose for electrophoresis supporting dielectric, electrophoresis 30min under 1 × TAE and 120V voltage, then dye 15min in 0.5 μ g/mL ethidium bromide staining liquid, observe with ultraviolet transilluminator, specific experiment the results are shown in Figure 4, Fig. 5 and Fig. 6, respectively corresponding diagram 1, Fig. 2 and Fig. 3.
Wherein, in Fig. 4, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 expression is cut the enzyme that commercially available RCA test kit rolling circle amplification product carries out; 0.5,2,4 be illustrated respectively in RCA reaction system add PEG 3350 solution of 0.5 μ l, 2 μ l and 4 μ l after the enzyme that carries out of the rolling circle amplification product that obtains cut, corresponding diagram 1.
In Fig. 5, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 expression is cut the enzyme that commercially available RCA test kit rolling circle amplification product carries out; 0.5,1,1.5 be illustrated respectively in RCA reaction solution add mPEG-SC 2000 solution of 0.5 μ l, 1 μ l and 1.5 μ l after the enzyme that carries out of the rolling circle amplification product that obtains cut, corresponding diagram 2.
In Fig. 6, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 expression is cut the enzyme that commercially available RCA test kit rolling circle amplification product carries out; 0.5,1.5,3 be illustrated respectively in this RCA reaction system add mPEG-SC 5000 solution of 0.5 μ l, 1.5 μ l and 3 μ l after the enzyme that carries out of the rolling circle amplification product that obtains cut, corresponding diagram 3.
From the result of Fig. 4, Fig. 5 and Fig. 6, after each RCA product enzyme is cut, not only stripe size (about 2686bp) is correct, and the brightness flop gradient of band is consistent with result corresponding in Fig. 1, Fig. 2 and Fig. 3, demonstrates the experimental result in embodiment 1.
Embodiment 3
Investigate PEG 3350, mPEG-SC 2000 and mPEG-SC 5000 respectively on the impact (taking mono-clonal as template) of RCA amplification efficiency
1, the structure of recombinant plasmid pUC19-hip: colibacillary hip gene will be derived from and insert in pUC19 plasmid, by connect transforms and use bacterium colony PCR, enzyme cuts and identifies and sequence verification, can obtain recombinant plasmid pUC19-hip;
2, transform: get 20ng recombinant plasmid pUC19-hip, be converted into 100 μ l E.coli BL21 competent cells, conversion is being cultivated containing on the LB flat board of 1 ‰ Ampicillin (100 μ g/ml), then the sub-fraction of the single bacterium colony of picking one is dissolved in the sterilized water of 50 μ l, mixing, obtains mono-clonal template;
3, with reference to the method for embodiment 1, PEG, mPEG-SC 2000, mPEG-SC 5000 is observed on the impact of RCA amplification efficiency;
4, RCA amplified production detects: after question response terminates, get 3 μ l RCA products with 1% sepharose for electrophoresis supporting dielectric, electrophoresis 30min under 1 × TAE and 120V voltage, then dye 15min in 0.5 μ g/mL ethidium bromide staining liquid, observe with ultraviolet transilluminator, specific experiment the results are shown in Figure 7, Fig. 8 and Fig. 9.
Described PEG 3350 solution add-on is 0.5 μ l, 1 μ l, 2 μ l or 4 μ l; Described mPEG-SC 2000 solution add-on is 0.5 μ l, 1 μ l, 1.5 μ l or 3 μ l; Described mPEG-SC 5000 solution add-on is 0.5 μ l, 1.5 μ l, 3 μ l or 4 μ l.
Method for expressing same Fig. 1, Fig. 2 and Fig. 3 respectively of each swimming lane of Fig. 7, Fig. 8 and Fig. 9.Wherein, in Fig. 7, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,1,2,4 PEG 3350 solution being illustrated respectively in reaction system the 0.1g/ml adding 0.5 μ l, 1 μ l, 2 μ l and 4 μ l.In Fig. 8, each swimming lane represents: M is 1kb DNA LadderMarker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,1,1.5,3 mPEG-SC 2000 solution being illustrated respectively in reaction solution the 0.2mg/l adding 0.5 μ l, 1 μ l, 1.5 μ l and 3 μ l.In Fig. 9, each swimming lane represents: M is 1kb DNA Ladder Marker; 0 is the rolling circle amplification product of commercially available RCA test kit; 0.5,1.5,3,4 mPEG-SC 5000 solution being illustrated respectively in reaction system the 0.1mg/l adding 0.5 μ l, 1.5 μ l, 3 μ l and 4 μ l.
As can be seen from Fig. 7, Fig. 8 and Fig. 9: when taking mono-clonal as template, along with the increase of PEG3350, the mPEG-SC 2000 added and mPEG-SC 5000 volume, DNA band gradually thicker, brighten, DNA rolling circle amplification efficiency significantly improves, reach a certain amount of after, the amplification of product starts to be suppressed.
Embodiment 4
The each RCA product (the monoclonal rolling circle amplification product containing pUC19-hip recombinant plasmid) embodiment 3 obtained carries out pcr analysis
Primer 1 (as shown in SEQ ID NO:1):
5′-ATGACCAAGTCAGAATTGATAG-3′；
Primer 2 (as shown in SEQ ID NO:2):
5′-TTAACCGTAAATATTGGCGCGATC-3′。
Comprise the following steps:
(1) according to the form below preparation PCR reaction solution 10 μ l:
| Reagent | Usage quantity (μ l) | Final concentration |
| 2×Phusion Master Mix | 5 | 1× |
| Primer 1 | 0.1 | 5pmol |
| Primer 2 | 0.1 | 5pmol |
| The RCA product of embodiment 3 | 0.2 | |
| Sterile purified water | 4.6 | |
| Cumulative volume | 10 |
(2) PCR reaction is carried out by following condition: 95 DEG C of 1min; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations; Last 72 DEG C of 5min.
(3) pcr amplification product detects: get 3 μ l PCR primer with 1.5% sepharose for electrophoresis supporting dielectric, electrophoresis 30min under 1 × TAE and 120V voltage, then dye 15min in 0.5 μ g/mL ethidium bromide staining liquid, observe with ultraviolet transilluminator, concrete electrophoresis result is shown in Figure 10, Figure 11 and Figure 12, respectively corresponding diagram 7, Fig. 8 and Fig. 9.Wherein, the pcr amplification product sequence of recombinant plasmid pUC19-hip is as shown in SEQ ID NO:4.
In Figure 10, each swimming lane represents: M is 100bp DNA Ladder Marker; 0 represents the PCR primer to commercially available RCA test kit rolling circle amplification product; 0.5,1,2,4 be illustrated respectively in RCA reaction system add PEG 3350 solution of 0.5 μ l, 1 μ l, 2 μ l and 4 μ l after the PCR primer of rolling circle amplification product that obtains, corresponding diagram 7.
In Figure 11, each swimming lane represents: M is 100bp DNA Ladder Marker; 0 represents the PCR primer to commercially available RCA test kit rolling circle amplification product; 0.5,1,1.5,3 be illustrated respectively in RCA reaction solution add mPEG-SC 2000 solution of 0.5 μ l, 1 μ l, 1.5 μ l and 3 μ l after the rolling circle amplification product that the obtains PCR primer of carrying out, corresponding diagram 8.
In Figure 12, each swimming lane represents: M is 100bp DNA Ladder Marker; 0 represents the PCR primer to commercially available RCA test kit rolling circle amplification product; 0.5,1.5,3,4 be illustrated respectively in this RCA reaction system add mPEG-SC 5000 solution of 0.5 μ l, 1.5 μ l, 3 μ l and 4 μ l after the PCR primer of rolling circle amplification product that obtains, corresponding diagram 9.
From the result of Figure 10, Figure 11 and Figure 12, after each RCA product is carried out PCR, PCR primer not only stripe size (about 285bp) is correct, and the brightness flop gradient of band is consistent with result corresponding in Fig. 7, Fig. 8 and Fig. 9, demonstrates the experimental result in embodiment 3.
The present invention by adding certain density PEG or derivatives thereof in RCA reaction system, the target template DNA be dispersed in system and random primer can be gathered together, improve the relative concentration of each component of RCA, add the pairing probability between target nucleic acid template and primer, thus the efficient amplification realized target nucleic acid sequence, largely solve the problems such as RCA technology speed of response is slow, sensitivity is low.In a word, the present invention can improve the efficiency of rolling circle amplification significantly, strengthens the sensitivity of rolling circle amplification, achieves the further optimization to commercially available RCA test kit.
Claims (4)
1. utilize poly-compounds to improve a method for RCA amplification efficiency, it is characterized in that: comprise the following steps:
(1) configuration concentration is mPEG-SC 5,000 three kinds of solution of the PEG 3350 of 0.1g/ml, the mPEG-SC 2000 of 0.2mg/l and 0.1mg/l respectively, for subsequent use;
(2) TempliPhi is used
tMdNA sequencing template amplification test kit:
A. sex change: using pUC 19 plasmid DNA as template, adds 1ng pUC 19 plasmid DNA in 5 μ l sample buffers, and after mixing, 95 DEG C of heating 3min, are then cooled to room temperature or 4 DEG C, obtain denaturing sample liquid;
B. cultivate: in 5 μ l reaction buffers, add 0.2 μ l Phi29DNA polysaccharase, mixing, is placed on ice, obtains premix synthase liquid; Add in described denaturing sample liquid by 5 μ l premix synthase liquid, mixing, obtains RCA reaction solution; PEG 3350 solution of preset vol, mPEG-SC 2000 solution, mPEG-SC 5000 solution is added respectively in each RCA reaction solution; Each RCA reaction solution is respectively at water-bath 4-18h at 30 DEG C; 65 DEG C of heating 10min, are cooled to 4 DEG C; 3 μ l RCA products are got 3 μ l reaction mixtures with 1% sepharose carry out electrophoresis in 1% sepharoses respectively.
2. a kind of method utilizing poly-compounds to improve RCA amplification efficiency as claimed in claim 1, it is characterized in that: in described step B, PEG 3350 solution add-on is 0.5 μ l, 2 μ l or 4 μ l.
3. a kind of method utilizing poly-compounds to improve RCA amplification efficiency as claimed in claim 1, it is characterized in that: in described step B, mPEG-SC 2000 solution add-on is 0.5 μ l, 1 μ l or 1.5 μ l.
4. a kind of method utilizing poly-compounds to improve RCA amplification efficiency as claimed in claim 1, it is characterized in that: in described step B, mPEG-SC 5000 solution add-on is 0.5 μ l, 1.5 μ l or 3 μ l.
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| CN105695571A (en) * | 2016-01-29 | 2016-06-22 | 苏州金唯智生物科技有限公司 | DNA quantitative method based on rolling circle amplification |
| CN105802956A (en) * | 2016-04-22 | 2016-07-27 | 华侨大学 | Method for improving RCA (Rolling Circle Amplification) efficiency by using polyethylenimine |
| CN107686860A (en) * | 2017-07-11 | 2018-02-13 | 华侨大学 | One kind improves more specific methods of primer RCA |
| WO2019129185A1 (en) * | 2017-12-28 | 2019-07-04 | 南京金斯瑞生物科技有限公司 | Method for rapidly preparing sanger sequencing template |
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| CN107686860A (en) * | 2017-07-11 | 2018-02-13 | 华侨大学 | One kind improves more specific methods of primer RCA |
| WO2019129185A1 (en) * | 2017-12-28 | 2019-07-04 | 南京金斯瑞生物科技有限公司 | Method for rapidly preparing sanger sequencing template |
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