WO2021104252A1 - Kit for single-cell whole-genome amplification and use thereof - Google Patents

Abstract

The present invention relates to a method and a kit for genome amplification and the use thereof. Disclosed is a method for single-cell whole-genome amplification, the method comprising the following steps: a. resuspending a single-cell sample to be amplified in an oil phase; b. lysing the cells, which are resuspended in the oil phase, obtained in step a; and c. introducing DNA polymerase, primers, and other reagents necessary for an amplification reaction into the sample obtained in step b so as to perform an amplification reaction. Also disclosed are an amplification kit, and the uses of the above-mentioned amplification method and the amplification kit. The disclosed amplification method and kit can reduce deviation caused during an amplification process, have a highly efficient and stable amplification effect, and can improve the genome coverage of single-cell amplification.

Description

Single cell whole genome amplification kit and its application Technical field

The invention relates to a genome amplification method, a kit and its application.

Background technique

The rapid development of DNA sequencing technology has enabled the academic community to have a more comprehensive understanding of the genomes of various species, including humans. The whole-genome amplification (WGA) of next-generation sequencing is used in biology and medicine. There are a wide range of applications. With the rapid development of sequencing technology, single cell separation technology and whole genome amplification technology, single cell research technology is listed as one of the most noteworthy technologies in the next few years. Sequencing the genome at the single-cell level can better solve the limitations of traditional technologies. The difficulty of single-cell whole-genome amplification technology lies in the isolation of single-cell and whole-genome amplification. Single-cell whole-genome amplification technology is the prerequisite for single-cell sequencing, and this technology can be used to reveal structural differences in single-cell genomes.

The common method of single-cell genome sequencing requires the use of DNA polymerase and high-throughput short-tailed DNA sequencing to perform extensive in vitro amplification of the genome of a single cell. These methods have two significant shortcomings. One is that polymerase replication errors may generate thousands of false positives, and the other is that relatively short sequence reads hardly contain any haplotype information. In addition, single-cell research technology has some problems, such as amplification bias, non-specific amplification, low sensitivity, poor reproducibility, high operating error rate, and foreign contamination. For example, MDA is currently recognized as the best single-cell genome amplification technology. It can perform high-fidelity and uniform amplification of the entire genome, amplifying fragments of 10 to 100 kb in size, and can provide a large number of uniform and complete genome sequences. However, MDA also has some shortcomings, especially the significant non-specific amplification, and there is still amplification preference [9].

Single cell isolation technology will also affect genome amplification. Studies have shown that in the process of single-cell Raman microspectroscopy analysis of bacterial cells, after direct laser irradiation, their membrane integrity will be lost, and even a large number of cells will die. The laser radiation during Raman analysis and sorting will affect the physiological activity of cells. Cause certain damage [10]. In addition, due to possible causes such as intracellular nucleic acid damage caused by laser radiation, the coverage of conventional bacterial single-cell sequencing after ejection generally does not exceed 20%, so genome assembly is relatively difficult [11,12]. In response to these problems, how to improve the accuracy of amplification, coverage and convenient operation is the research direction of future scientific researchers.

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2. Schroder UC, Belites C, Assmann C, Glaser U, Hubner U, Pfister W, et al. Detection of vancomycin resistances in enterococci within 3 1/2 hours. Sci Rep-Uk 2015; 5.

3.Teng L, Wang X, Wang X, Gou H, Ren L, Wang T, et al. Label-free, rapid and quantitative phenotyping of stress response in E. coli via ramanome. Sci Rep 2016; 6:34359.

4. Gruber-Vodicka HR, Dirks U, Leisch N, Baranyi C, Stoecker K, Bulgheresi S, et al. Paracatenula, an ancient symbiosis between thiotrophic Alphaproteobacteria and catenulid (USA 29)worms. P Natl 29 (USA) 78 worms. -12083.

5. Briers Y, Staubli T, Schmid MC, Wagner M, Schuppler M, Loessner MJ. Intracellular Vesicles as Reproduction Elements in Cell Wall-Deficient L-Form Bacteria. Plos One 2012; 7(6).

6. Majed N, Chernenko T, Diem M, Gu AZ.Identification of Functionally Relevant Populations in Enhanced Biological Phosphorus Removal Processes Based On Intracellular Polymers Profiles and Insights Into the Diversity 10; Technological Heterogenity 46; -5017.

7.Milucka J, Ferdelman TG, Polerecky L, Franzke D, Wegener G, Schmid M, et al. Zero-valent sulphur is a key intermediate in marine methane oxidation. Nature 2012; 491(7425): 541-+.

8.Li MQ, Canniffe DP, Jackson PJ, Davison PA, FitzGerald S, Dickman MJ, et al. Rapid resonance to probe carbon dioxide fixation by single cells in microbial communities.Isme: 875 885

9 Woyke, T., D.F.R. Doud, and F. Schulz, The trajectory of microbial single-cell sequencing. Nat Methods, 2017. 14(11): p.1045-1054.

10 Mathey, R., et al., Viability of 3h grown bacterial micro-colonies after direct Raman identification. J Microbiol Methods, 2015.109: p. 67-73.

11 Jing X, Gou H, Gong Y, et al. Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean. Environment Microbiology, 2018, 4: 29727057.

12 Song Y, Kaster A K, Vollmers J, et al. Single-cell genomics based on Raman sorting reveals novel carotenoid-containing bacteria in the Red Sea. Microbal Biotechnology, 2017, 10(1): 125-137.

13 Yuan X, Song Y, Song Y, et al. Effect of laser irradiation on cell function and its implications in Raman Spectroscopy. Applied and Environmental Microbiology, 2018, 84(8): e02508-17.

Summary of the invention

The invention provides a method for amplifying the whole genome of a single cell, a kit and the application of the amplification method.

In one aspect, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained from step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, DNA polymerase, primers, and other necessary reagents for the amplification reaction are provided for the amplification reaction.

The method is applicable to single-cell samples of all species, for example, bacteria (gram-positive bacteria and gram-negative bacteria), plant cells, animal cells, etc.

The single cell sample includes, but is not limited to, cells resuspended in PBS, cells resuspended in water, cells resuspended in medium, cells fixed with acetaldehyde, and single cells directly isolated.

The other reagents necessary for the amplification reaction include DTT, DMSO, 10*phi29 buffer, dNTP, H2O, etc.

The DNA polymerase described in the present invention includes but is not limited to vent DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, DNA polymerase I, (Klenow) large fragment, DNA polymerase I (E. coli), Sulfolobus DNA polymerase IV, phi29 DNA polymerase, Bst DNA polymerase, Equiphi29 DNA polymerase, etc., preferably Phi29 DNA polymerase.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, provide DNA polymerase, primers, and other necessary reagents for the amplification reaction. The oil phase includes one or more of mineral oil, fluorocarbon oil, and silane oil. Preferably, the oil phase is mineral oil.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, DNA polymerase, primers, and other necessary reagents are provided for the amplification reaction. The oil phase includes one or more of mineral oil, fluorocarbon oil, and silane oil. The oil phase SCC buffer O mainly includes mineral oil (light mineral oil, heavy mineral oil, etc.), fluorocarbon oil, and silanes. The oil phase also contains a surfactant. Preferably, the surfactant is span-80 or EM90.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, DNA polymerase, primers, and other necessary reagents are provided for the amplification reaction. The mixing ratio of the single cell sample and the oil phase is 1:0.5 to 1:20; preferably, the mixing ratio of the single cell sample and the oil phase is 1:1 to 1:10; more preferably, the mixing ratio of the single cell sample and the oil phase is 1:1 to 1:2. The oil phase includes one or more of mineral oil, fluorocarbon oil, and silane oil. Preferably, the oil phase is mineral oil.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, provide DNA polymerase, primers, and other necessary reagents for the amplification reaction. The single cell sample to be amplified in a is a single cell suspended in water or PBS buffer.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, DNA polymerase, primers, and other necessary reagents are provided for the amplification reaction. The single cell sample is a single cell obtained by laser radiation sorting, preferably, a single cell obtained by Raman signal sorting.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, provide DNA polymerase, primers, and other necessary reagents for the amplification reaction. It is characterized in that, in step b, the method for lysing the cells is selected from any one of chemical lysis, enzymatic lysis, or mechanical lysis or any combination thereof.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a resuspend the single-cell sample to be amplified in an oil phase; b. Cells suspended in the oil phase are lysed; c. In the sample obtained in step b above, DNA polymerase, primers, and other necessary reagents are provided for the amplification reaction. It is characterized in that, in step b, the cell is lysed by chemical lysis, preferably alkaline lysis is used. More preferably, the alkaline lysis solution (Lysis buffer A) is composed of Lysis buffer A1 and DTT; the Lysis buffer A1 is composed of a solute and a solvent; the solvent is water; the solute and its concentration are: 0.09-0.11mM KOH and 0.9 -1.1mM EDTA; preferably, the Lysis buffer A1 is composed of a solute and a solvent; the solvent is water; the solute and its concentration are: 0.1mM KOH and 1mM EDTA; the DTT is composed of a solute and a solvent; the solvent is water; solute Its concentration is: 0.1-5M DTT.

On the other hand, the present invention provides a single-cell whole-genome amplification method, which is characterized by comprising the following steps: a. Resuspend the single-cell sample to be amplified in an oil phase; b. Obtained in step a above Cells resuspended in the oil phase are lysed; c. In the sample obtained in step b above, provide DNA polymerase, primers, and other necessary reagents for the amplification reaction. In step c, the DNA polymerase is Phi29 DNA polymerase, or Equiphi29 (Thermo Fisher).

The present invention also provides a single-cell whole-genome amplification kit, which is characterized in that the kit includes a pretreatment solution for single-cell sample amplification, DNA polymerase, amplification buffer, and primers; the single-cell The pretreatment liquid before sample amplification is oil. Preferably, the DNA polymerase is Phi29 DNA polymerase, or Equiphi29 (Thermo Fisher).

In another aspect, the present invention provides a single-cell whole-genome amplification kit, characterized in that the kit includes a pre-treatment solution for single-cell sample amplification, DNA polymerase, amplification buffer, and primers; The pretreatment liquid before single cell sample amplification is an oil phase. Preferably, the oil in the sample pretreatment liquid before single cell sample amplification is selected from one or more of mineral oil, fluorocarbon oil, and silane oil .

Preferably, the pretreatment solution before single-cell sample expansion further contains a surfactant. Preferably, the surfactant is span-80 or EM90.

Preferably, the kit also contains instructions.

The present invention also protects another single-cell genome amplification method, which includes the following steps:

(1) Put 0.5μl of cell material (resuspended in PBS) into a microcentrifuge tube, add 1μl of oil solution SCC buffer O, shake and mix thoroughly;

The oil solution SCC buffer O solution contains one or more of mineral oil, fluorocarbon oil, and silane oil;

(2) Add 1μl of denatured lysate Lysis buffer A to the solution obtained in step (1), shake and mix thoroughly, centrifuge briefly, and then incubate at 65°C for 10-15min;

The denatured lysis solution Lysis buffer A is composed of Lysis buffer A1 and DTT;

The Lysis buffer A1 is composed of a solute and a solvent; the solvent is water; the solute and its concentration are: 0.09-0.11mM KOH and 0.9-1.1mM EDTA;

The DTT is composed of a solute and a solvent; the solvent is water; the solute and its concentration are: 0.1-5M DTT;

(3) Add 1μl of Neutralization buffer B to the solution obtained in step (2), shake and mix thoroughly, and centrifuge briefly to obtain a template solution, and store on ice;

The neutralization buffer B is composed of a solute and a solvent; the solvent is water; the solute and its concentration are: 54-66mM KH2PO4 and 4.5-5.5mM K2HPO4;

(4) Take 3 μL of the template solution obtained in step (3), add 10 μL of the reaction mixture, incubate at 30°C for 8 hours, and then stand at 65°C for 10 minutes to obtain the amplified product;

The reaction mixture is composed of 2.25 μl water, 7.25 μl SCC-reaction buffer C ₁ solution and 0.5 μl DNA polymerase;

The SCC-reaction buffer C1 solution consists of 1 μl 10×Phi29 buffer, 4.54 μl water, 0.75 μl DMSO, and 0.96 μl amplification system A;

A single amplification system A consists of 0.06μl dNTP, 0.75μl N6primers and 0.15μl DTT; the N6primers are random primers composed of 6 deoxyribonucleotides, A, G, C and T are randomly arranged, and the 3′ end Two kinds of nucleotides are modified with phosphosulfate;

In the dNTP solution, the concentration of dATP/dTTP/dCTP/dGTP is 10 mM; in the N6primers solution, the total primer concentration is 50 μM;

The DNA polymerase is 0.5 μL Phi29 DNA polymerase;

The concentration of the Phi29 DNA polymerase is 10 U/μL.

The present invention also provides the application of any of the above methods in genome sequencing, holographic array, quantitative PCR and the like. Preferably, genome sequencing is second-generation sequencing.

The present invention also provides the application of any of the above kits in genome sequencing, holographic array, quantitative PCR and the like. Preferably, genome sequencing is second-generation sequencing.

The kit provided by the present invention can be used for single cell samples of all species, for example, bacteria (Gram-positive bacteria and Gram-negative bacteria), sorted or tissue cultured animal cells, plant cells, etc. The kit can be applied to the functional research of complex microbial communities and bacterial infections, and has great application prospects and promotion value.

In the first aspect, the present invention provides a single-cell whole-genome amplification method that can improve genome coverage. The method performs high-fidelity and high-uniform amplification of different sites in the whole genome of a single cell, and the amplified products can be used for high-throughput sequencing analysis.

In the second aspect, the present invention provides kits for single cell amplification, existing amplification methods and single cell kits (commonly used Qiagen single cell kit), and genome coverage obtained by single cell amplification after Raman measurement The rate is relatively low. This kit can significantly reduce the deviation caused by the amplification process, and has a highly efficient and stable amplification effect, which can significantly increase the genome coverage of single-cell amplification, and can provide more than 90% of the genome from a single cell DNA sequence information.

Detailed ways

The following specific examples are further descriptions of the methods and technical solutions provided by the present invention, but should not be construed as limiting the present invention.

Example 1. Preparation of reagents

1. Prepare sufficient lysis buffer (Lysis buffer A) for the single-cell genome amplification reaction process (Table 1);

Table 1 Preparation of Lysis buffer A: Lysis buffer A1 is 0.1mM KOH, 1mM EDTA, mix in the following proportions when using

Component	Amount
Lysis buffer A1	33μl
DTT	3μl
Total	36μl

2. Preparation before the experiment

Set up a 65℃ water bath or metal bath heater;

3. The temperature of the thermal lid of the PCR machine is set to 70°C.

Example 2. Amplification of E. coli cells

1. 0.5μl of E. coli cell material (the cell material includes but not limited to cells resuspended in PBS, cells resuspended in water, cells resuspended in culture medium, cells fixed with acetaldehyde, single cells directly isolated, etc.) Put it in a microcentrifuge tube, add 0.5 to 20 times the sample volume of mineral oil (1 times is better), shake and mix thoroughly.

2. Add the same volume of Lysis buffer A as the oil phase pretreatment sample in step 1 above, shake and mix thoroughly, and centrifuge briefly. Enzymatic lysis, such as lysozyme lysis, has the same effect of mechanical lysis and chemical lysis. The enzymatic lysis includes but is not limited to SDS and lysozyme, the mechanical lysis includes heat shock and ultrasonic lysis, and the working method of the ultrasonic lysis is at a lysis power of 300-400w, each time it works intermittently after 2-5s 4～10s, total working time is 6～10min. The chemical lysis solution includes but is not limited to any of the following: 0.1mM KOH, 1mM EDTA, 1M DTT; 50mM KAc, 20mM Tris-Ac, 10mM MgAc, 0.2% Trition X-100; 1xPBS, 1xThermopolbuffer, 30mM Tris- HCl; 10mM Tris-HCl, 25mM EDTA, 100mM NaCl; 0.5% SDS, 10mM Tris-HCl, 50mM KCl; 10mM Tris-HCl, 100mM EDTA, 0.5% SDS; 30mM Tris-HCl, 10mM EDTA, 1% SDS; 30mM Tris-HCl, 2mM KCl, 0.2% Triton X-100; Urea, iodoacetamide and NH4HCO3. ) The best is 0.1mM KOH, 1mM EDTA, 1M DTT (PH=7-9).

3. Incubate at 65°C for 10-15min.

4. Add Neutralization buffer B with the same volume as Lysis buffer. Neutralization buffer B is configured by 60mM KH2PO4 and 5mM K2HPO4. Shake well and mix well, centrifuge briefly, and store on ice.

5. Prepare the amplification mixture. Mix and centrifuge briefly;

DNA polymerases include but are not limited to vent DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, DNA polymerase I, (Klenow) large fragments, DNA polymerase I (E.coli), Sulfolobus DNA polymerase IV, phi29 DNA polymerase, Bst DNA polymerase, Equiphi29 DNA polymerase, etc., the best is Phi29 DNA polymerase. Keep on ice for use; other reagents need to be dissolved at room temperature before shaking and centrifuge, and placed on ice for later use; ddH ₂ O at room temperature for use; SCC-reaction buffer C1 solution can be 10% by volume 10× It consists of Phi29buffer, water, 7.5% DMSO by volume, 10mM dNTP, N6primers with a total primer concentration of 50μM, and 1M DTT. The amplification mixture can be prepared according to Table 2. After water (ddH ₂ O), SCC-reaction buffer C ₁ , shake and mix well and centrifuge, then add Phi29 DNA polymerase.

Table 2 Preparation of amplification mixture

6. Add 10 μl reaction mixture to each reaction (3 μl denatured DNA, step 5).

7. Incubate at 30°C for 8 hours.

8. Inactivate Phi29 DNA polymerase activity at 65°C for 10 min.

9. Dilute the amplified DNA 20 times, and take 1μl/reaction as a template for specific PCR detection and analysis.

10. Purify and sequence the successfully amplified MDA product.

11. For PCR analysis, the amplified DNA was diluted 20 times, and 1 μl of the reaction was used as a template.

In order to explore the effect of oil phase pretreatment on DNA amplification of Escherichia coli cells before amplification by adding samples, 8 samples obtained by amplifying samples with the same volume of oil phase pretreatment were used, and the samples were not pretreated with oil phase. According to the standard procedure, the 8 samples of the sample were amplified and compared, and each sample contained 1 E. coli cell. According to 1% agarose gel electrophoresis analysis of MDA and PCR amplification products, it was found that 8 samples of the oil phase treated samples were all positive (that is, 1% gel electrophoresis detected typical MDA and PCR bands) , While only 5 samples amplified by the standard method were positive. In addition, the bacterial gene fragments were successfully amplified in the samples pre-treated with the oil phase, and no contamination was found. The MDA reaction yielded 732-3,600ng product, which is sufficient for subsequent next-generation sequencing preparation. Each selected 5 successfully amplified samples to construct a library and sequenced with Illumina HiSeq2500. Through assembly, 10 single amplified genomes were obtained (5 contained oily phase when amplified and 5 did not contain) (Table 3). The genome coverage rate obtained under the standard amplification conditions of the oil-free pretreatment sample was 49.82%-52.60%, while the genome coverage rate of the sample amplified by the oil-phase pretreatment sample was significantly higher, 89.78% to 99.97% .

Table 3. Assembly and evaluation of E. coli single-cell genome

Example 4 Saccharomyces cerevisiae cell expansion

1. Put 0.5μl of Saccharomyces cerevisiae cell material (resuspended in PBS) into a microcentrifuge tube, add 0.5μl of mineral oil and shake well to mix.

2. Add 1μl Lysis buffer A, shake and mix thoroughly, and centrifuge briefly.

3. Incubate at 65°C for 10-15min.

4. Add 1μl Neutralization buffer B. Shake well and mix well, centrifuge briefly, and store on ice.

5. Prepare the amplification mixture according to Table 4. Mix and centrifuge briefly;

Phi29DNA polymerase is placed on ice for use; other reagents need to be dissolved at room temperature before being shaken and centrifuged, and placed on ice for use; ddH ₂ O is placed at room temperature for use; the amplification mixture can be prepared according to Table 4. After water (ddH ₂ O), SCC-reaction buffer C ₁ , shake and mix well and centrifuge, then add Phi29 DNA polymerase.

Table 4 Preparation of amplification mixture

②Prepare multiple reaction mixtures and prepare an additional 10% according to the number of reactions.

6. Add 10 μl reaction mixture to each reaction (3 μl denatured DNA, step 5).

7. Incubate at 30°C for 8 hours.

8. Inactivate the activity of Phi29 DNA polymerase at 65°C for 10 minutes.

9. Dilute the amplified DNA 20 times, and take 1μl/reaction as a template for specific PCR detection and analysis.

10. Purify and sequence the successfully amplified MDA product.

11. For PCR analysis, the amplified DNA was diluted 20 times, and 1 μl of the reaction was used as a template.

In order to explore the effect of adding oil-phase pretreatment samples on the DNA amplification of Saccharomyces cerevisiae cells, 8 pretreated Saccharomyces cerevisiae cell samples and the other 8 samples without oil phase pretreatment were amplified according to standard procedures. Each sample contains 1 E. coli cell. According to 1% agarose gel electrophoresis analysis of MDA and PCR amplification products, 8 samples of the samples pretreated with oil phase were positive (that is, 1% gel electrophoresis detected typical MDA and PCR bands) , While the samples that were not pre-treated with the oil phase were amplified by standard methods, only 7 samples were positive. In addition, the bacterial gene fragments were successfully amplified in the oil-phase pretreated samples, and no contamination was found. The MDA reaction yielded 998-4,020ng product, which is enough for subsequent next-generation sequencing preparation. Next, the library was constructed using 5 samples pretreated with oil phase and 5 samples without oil phase pretreatment for amplification and sequencing with Illumina HiSeq2500. Through assembly, 10 single amplified genomes were obtained (Table 5). The genome coverage of samples without oil phase pretreatment under standard amplification conditions is 55.67%-67.80%, while the genome coverage of samples amplified by oil phase pretreatment samples is significantly higher, reaching 93.19%~ 99.89%.

Table 5. Assembly and evaluation of single cell genome of Saccharomyces cerevisiae cells

Example 5, Urine sample cell expansion

1. Put 0.5μl of urine sample cell material (resuspended in PBS) into a microcentrifuge tube, add 0.5μl of mineral oil and shake well to mix.

2. Add 1μl Lysis buffer A, shake and mix thoroughly, and centrifuge briefly.

3. Incubate at 65°C for 10-15min.

4. Add 1μl Neutralization buffer B. Shake well and mix well, centrifuge briefly, and store on ice.

5. Melt Phi29 DNA polymerase on ice. Dissolve other reagents at room temperature, shake to mix and centrifuge briefly.

6. Add 10 μl reaction mixture to each reaction (3 μl denatured DNA, step 5).

7. Incubate at 30°C for 8 hours.

8. Inactivate the activity of Phi29 DNA polymerase at 65°C for 10 minutes.

9. Dilute the amplified DNA 20 times, and take 1μl/reaction as a template for specific PCR detection and analysis.

10. Purify and sequence the successfully amplified MDA product.

11. For PCR analysis, the amplified DNA was diluted 20 times, and 1 μl of the reaction was used as a template.

In order to explore the effect of adding oil phase pretreatment samples on the DNA amplification of urine samples, 8 samples were amplified in the presence of 1 μl oil phase, and the other 8 samples were amplified according to standard procedures, and each sample contained 1 E. coli cells. According to 1% agarose gel electrophoresis analysis of MDA and PCR amplification products, it was found that 8 samples of the oil phase added samples were positive (that is, 1% gel electrophoresis detected typical MDA and PCR bands). Only 6 samples amplified by the standard method were positive. Bacterial gene fragments were successfully amplified in these samples, and no contamination was found. The MDA reaction yielded 805-3,430ng product, which was sufficient for subsequent preparation of next-generation sequencing. Next, the library was constructed and sequenced with Illumina HiSeq2500. Through assembly, 10 single amplified genomes were obtained (5 contained oily phase when amplified, 5 did not contain) (Table 6). The genome coverage rate obtained under standard amplification conditions without oil phase was 49.02%-57.60%, while the genome coverage rate obtained from the oily phase amplified samples was significantly higher, ranging from 92.18% to 99.57%.

Example 6. Urine sample cell expansion

Except that in step 1, the mineral oil is replaced with fluorocarbon oil, the other steps are the same as in Example 5. The results showed that the genome coverage of the oil-containing amplified samples was significantly higher than that of the control.

Table 6. Assembly and evaluation of single cell genome of urine samples

Claims (13)

1. A method for single-cell genome amplification, which is characterized by comprising the following steps:

   a. Resuspend the single cell sample to be amplified in the oil phase;

   b. Lyse the cells resuspended in the oil phase obtained in step a above;

   c. In the sample obtained in step b above, provide DNA polymerase, primers, and other necessary reagents for the amplification reaction to perform the amplification reaction.
2. The method according to claim 1, wherein the oil phase is selected from any one or any combination of mineral oil, fluorocarbon oil, and silane oil.
3. The method of claim 2, wherein the oil phase further comprises a surfactant, preferably, the surfactant is span-80 or EM90.
4. The method of claim 1, wherein the mixing ratio of the single cell sample and the oil phase is 1:0.5 to 1:20.
5. The method of claim 1, wherein the single-cell sample is a sample obtained by laser radiation sorting.
6. The method of claim 4, wherein the laser radiation sorting is Raman signal sorting.
7. The method of claim 1, wherein in step b, the cleavage is selected from any one of chemical cleavage, enzymatic cleavage, or mechanical cleavage, or any combination thereof.
8. The method of claim 6, wherein the chemical lysis is alkaline lysis.
9. The method of claim 1, wherein the DNA polymerase in step c is Phi29 DNA polymerase.
10. A kit for single-cell genome amplification, characterized in that the kit includes: a pretreatment solution before amplification of a single-cell sample, a DNA polymerase, an amplification buffer, and primers; the single-cell sample amplification The pretreatment liquid before the increase is the oil phase.
11. A kit according to 10, wherein the oil phase is selected from any one or any combination of mineral oil, fluorocarbon oil, and silane oil.
12. The application of the method according to any one of claims 1 to 9 in genome sequencing, holographic array, and quantitative PCR.
13. The application of the kit according to any one of claims 10 to 12 in sequencing, holographic array, and quantitative PCR.