Method for carrying out whole genome amplification by taking cell culture fluid as template

Abstract

The invention provides a method for carrying out whole genome amplification by taking cell culture fluid as a template, the whole genome amplification is carried out by simulating different cell culture fluids, an optimized whole genome amplification reagent and program are determined, and effective amplification of various culture fluid cells or trace genome templates is realized. Whole genome amplification products are subjected to quality inspection, database establishment and machine loading, and off-machine data are analyzed, so that the effectiveness and the accuracy of whole genome amplification data obtained by the method disclosed by the invention are proved. The problem that an amplification reagent on the current market cannot resist inhibition amplification is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of whole genome amplification, and in particular to a method for whole genome amplification using cell culture fluid as a template. Background Art

[0002] Each cell is unique. Even single cells from the same source differ from each other in many ways due to random biological processes and environmental disturbances. The second-generation sequencing technology is designed for cell populations, and the sample amount required is at the ng level. However, the DNA in a single cell (only about 6pg DNA) cannot meet the sample amount for sequencing, so it cannot meet the needs of scientific researchers for single-cell research. In order to make a small amount of samples meet the requirements of sequencing, whole genome amplification technology (WGA) was born. Whole genome amplification technology can evenly amplify very small amounts of DNA to obtain a large amount of DNA, so it is considered to be a basic method to solve this problem. It has been widely used in forensic medicine, diagnosis of single-gene genetic diseases, and analysis of disease genes, and has achieved good results.

[0003] Whole genome amplification is a technique for non-selective amplification of all genome sequences. Its main purpose is to maximize the amount of DNA on the basis of faithfully reflecting the whole picture of the genome, and to amplify the entire genome DNA of trace tissues and single cells without sequence bias, so as to provide sufficient DNA templates for subsequent multi-gene, multi-site analysis and comprehensive research on the genome. It is widely used in research on cancer research, embryonic development, assisted reproduction, cell differentiation, immune mechanism, microorganisms, etc. According to the different primers and amplification conditions, the methods of whole genome amplification are divided into the following: ligation-mediated PCR (LM-PCR), multiple displacement amplification (MDA), degenerate oligonucleotide primer PCR (DOP-PCR), multiple annealing loop amplification technology (MALBAC) and primer extension pre-amplification PCR (PEP-PCR).

[0004] Each whole genome amplification method has its own advantages, disadvantages and application directions, but the starting template is a trace amount of gDNA or a single cell, which has high requirements on template quality, requires a complete genome or cell, and the cell culture medium and buffer should not contain Ca 2+ and Mg 2+ , so as not to affect the single cell amplification experiment. However, it is difficult to achieve no interference from any other substances for the cells separated or cultured by customers, or trace genomic templates such as forensic cases. Therefore, an amplification reagent with obvious anti-inhibition effect is urgently needed, which can realize whole genome amplification using various cell culture fluids as templates. Summary of the invention

[0005] In view of this, the present invention provides a method for amplifying a variety of culture fluid cells or trace genome templates. The method of the present invention can use cell culture fluid as a template to perform whole genome amplification with resistance to inhibition.

[0006] A first aspect of the present invention provides a method for whole genome amplification, the method comprising the steps of:

[0007] S1. Provide a sample containing a trace amount of genomic DNA as a template;

[0008] S2. Pre-amplify the template in the sample using a degenerate oligonucleotide primer to obtain a pre-amplification product, wherein the pre-amplification reaction procedure includes:

[0009] Denature at 95°C-98°C for 1-5 min; pre-denature at 95°C-98°C for 10-30 s, incubate at the first temperature for 40-60 s, incubate at the second temperature for 30-50 s, incubate at the third temperature for 20-40 s, incubate at the fourth temperature for 30-50 s, incubate at the fifth temperature for 30-50 s, for a total of 10-15 cycles;

[0010] Wherein, the first temperature is 10°C-20°C, the second temperature is 5°C-15°C higher than the first temperature, the third temperature is 5°C-15°C higher than the second temperature, the fourth temperature is 20°C-40°C higher than the third temperature, the fifth temperature is 5°C-15°C higher than the fourth temperature, and the fifth temperature is not higher than 80°C;

[0011] S3. Amplify the pre-amplification product using degenerate oligonucleotide primers.

[0012] In another preferred embodiment, the reaction procedure of the pre-amplification in step S2 includes:

[0013] Denature at 95°C for 2 min; pre-denaturation at 95°C for 15 s, incubate at 15°C for 50 s, incubate at 25°C for 40 s, incubate at 35°C for 30 s, incubate at 65°C for 40 s, and incubate at 75°C for 40 s, for a total of 10-15 cycles.

[0014] In another preferred embodiment, the sample in step S1 is cell culture fluid.

[0015] In another preferred embodiment, the sample in step S1 contains cell culture medium or embryo culture medium.

[0016] In another preferred embodiment, the cell culture medium includes commercial cell culture media such as 1640 and DMEM; the embryo culture medium includes embryo culture media such as Vitrolife G-2 and SAGE ART.

[0017] In another preferred example, the sample contains genomic DNA from 1 to 10 cells.

[0018] In another preferred example, the sample contains 5-30 pg / ul gDNA.

[0019] In another preferred embodiment, in step S1, a cell culture medium containing 1-10 cells is treated with a cell lysis enzyme to obtain a template containing a trace amount of genomic DNA.

[0020] In another preferred embodiment, the cell lytic enzyme comprises one or more of proteinase K, QIAGEN Protease, pepsin, trypsin, lysozyme, etc.; preferably proteinase K.

[0021] In another preferred embodiment, the pre-amplification reaction system for pre-amplification in step S2 comprises a pre-amplification reagent and a pre-amplification buffer, wherein the pre-amplification reagent comprises a DNA polymerase, a degenerate oligonucleotide primer and a dNTP; and the pre-amplification buffer comprises Tris-Cl, KCl, BSA, MgSO4, DMSO, and Triton X-100.

[0022] In another preferred embodiment, the pre-amplification reaction system comprises Tris-Cl 15-45 mM, KCl 15-45 mM, BSA 0.1-1 mg / ml; MgSO4 0.5-3 mM, DMSO 0.1%-10% (v / v) and Triton X-100 0.05-2% (v / v).

[0023] In another preferred embodiment, the reaction procedure for amplification in step S3 includes:

[0024] Denature at 95℃-98℃ for 1-5min; pre-denaturation at 95℃-98℃ for 10-30s, incubate at 60℃-70℃ for 30-120s, incubate at 70℃-80℃ for 30-120s, for a total of 10-20 cycles.

[0025] In another preferred embodiment, the reaction procedure for amplification in step S3 includes:

[0026] Denature at 95°C for 2 min; pre-denaturation at 95°C for 15 s, incubate at 65°C for 60 s, and incubate at 75°C for 60 s, for a total of 10-20 cycles.

[0027] In another preferred embodiment, the amplification reaction system for amplification in step S3 comprises an amplification reagent and an amplification buffer, wherein the amplification reagent comprises a DNA polymerase, a degenerate oligonucleotide primer and a dNTP; and the amplification buffer comprises Tris-Cl, KCl, BSA, MgSO4, DMSO, and Triton X-100.

[0028] In another preferred embodiment, the amplification reaction system comprises Tris-Cl 15-45 mM, KCl 15-45 mM, BSA 0.1-1 mg / ml; MgSO4 0.5-3 mM, DMSO 0.1%-10% (v / v) and Triton X-100 0.05-2% (v / v).

[0029] In another preferred embodiment, the DNA polymerase is selected from the following group: Phi29 DNA polymerase, Bst DNA polymerase, Vent polymerase, Deep Vent polymerase, Klenow Fragment DNA polymerase I, super-fidelity DNA polymerase, Taq polymerase, E. coli DNA polymerase, Long Amp Taq DNA polymerase and One Taq DNA polymerase; preferably Phi29 DNA polymerase.

[0030] In another preferred embodiment, the genomic DNA is eukaryotic genomic DNA, such as mammalian (eg human) genomic DNA.

[0031] In another preferred embodiment, the method is a non-diagnostic method, for example, in vitro cell cultures can be tested to obtain genomic information required for scientific research.

[0032] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the electrophoresis diagram of the whole genome amplification product of the simulated sample;

[0034] Figure 2 To simulate the analysis of samples, from top to bottom are cell gDNA control, single cell Takara product, and single cell amplification product of the method of the present invention;

[0035] Figure 3 This is the electrophoresis diagram of the whole genome amplification product of the culture fluid simulation sample containing trace amounts of gDNA;

[0036] Figure 4 This is a graph of the analysis of a culture fluid simulation sample containing trace amounts of gDNA, from top to bottom: 15pg gDNA Takara product of cells, and 15pg gDNA amplification product of the present invention. DETAILED DESCRIPTION

[0037] The present invention simulates different cell culture fluids for whole genome amplification, determines the optimized whole genome amplification reagents and procedures, and realizes the effective amplification of multiple culture fluid cells or trace genome templates. The whole genome amplification product is inspected, the library is built, the data is uploaded to the machine, and the data off the machine are analyzed, which proves the effectiveness and accuracy of the whole genome amplification data performed by the method of the present invention. The problem that the current amplification reagents on the market cannot resist inhibition of amplification is solved. Therefore, the method of the present invention can use cell culture fluid as a template to resist inhibition for whole genome amplification. On this basis, the inventors completed the present invention.

[0038] Different culture medium templates are simulated to obtain templates containing trace cells or trace genomic DNA; trace cell culture medium templates are mainly obtained by counting culture cells, diluting and plating, and using a micromanipulator pipette to draw 1-10 cells and put them into the corresponding culture medium to obtain the corresponding template; trace genomic DNA templates are mainly obtained by accurately quantifying the complete gDNA through the human genome quantification kit, and the quantified genome is diluted with cell culture medium to, for example, 15pg / ul, to form a culture medium simulation template containing trace gDNA.

[0039] The pre-amplification reaction system of the present invention can be set as one of a 15ul system, a 20ul system, and a 30ul system. The pre-amplification reaction procedure is mainly: denaturation at 95°C-98°C for 2min; pre-denaturation at 95°C-98°C for 15s, and gradient annealing extension for 30-50s (10-15cycles).

[0040] Advantages of the present invention include:

[0041] 1. The method of the present invention can achieve anti-inhibition amplification of whole genome amplification;

[0042] 2. The present invention can achieve whole genome amplification using various culture fluids (including cells or genomic DNA) as templates by optimizing the pre-amplification reaction procedure, pre-amplification reaction system, amplification reaction procedure and amplification reaction system, and the amplification product can meet the requirements of the machine.

[0043] 3. The method of the present invention can be widely used in forensic trace genome template amplification and blastocyst culture medium amplification, which reduces the sample delivery requirements and can achieve whole genome amplification of single cells or trace genomes containing culture medium.

[0044] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Materials and reagents used in the present invention are commercially available products unless otherwise specified.

[0045] Example 1. Comparison of whole genome amplification of cells in simulated cell culture medium

[0046] 1. Preparation of simulated culture medium

[0047] 1. Count the cells using cultured 293T cells to determine the number of cells, plate and dilute them into 96-well culture dishes to ensure that each well contains 1-5 cells. Use the mouth pipette method to take samples under a microscope and pipette the cells into DMEM culture medium to complete the preparation of the cell template dilution solution;

[0048] 2. Whole Genome Amplification

[0049] 1) Sample lysis: Take 9.8 μl of culture medium sample, add 0.2 μl of cell lysis enzyme (lysis enzyme is proteinase K, which can be purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW2572L), shake and mix, the reaction procedure is as follows.

[0050] Table 1

[0051]

[0052] 2) Pre-amplification reaction

[0053] Degenerate oligonucleotide primer PCR is used to amplify a variety of culture cells or trace genomic templates.

[0054] The pre-amplification reagents included: DNA polymerase (from Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW2608) 2U / μl, degenerate oligonucleotide primer (5'-CCGACTCGAGNNNNNNATGTGG-3', SEQ ID No. 1) 4mmol / L; dNTP: 0.5mmol / L.

[0055] The pre-amplification buffer contained: Tris-Cl 45 mM, KCl 45 mM, BSA 1 mg / ml; MgSO4 3 mM, DMSO 0.5% and Triton X-100 0.5%.

[0056] The pre-amplification reaction system is 30ul.

[0057] According to the number of reactions N, mix equal volumes of pre-amplification buffer and pre-amplification reagent, vortex to mix, centrifuge briefly, and obtain pre-amplification mixture for use; add 20 μl of pre-amplification mixture to 10 μl of cleavage reaction product in the previous step, and run the following program.

[0058] Table 2

[0059]

[0060]

[0061] 3) Amplification reaction

[0062] The amplification reagents included: DNA polymerase (available from Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW0938M) 2U / μl, degenerate oligonucleotide primer (5'-CCGACTCGAGNNNNNNATGTGG-3', SEQ ID No. 1) 4mmol / L; dNTP: 0.5mmol / L.

[0063] The amplification buffer contained: Tris-Cl 45 mM, KCl 45 mM, BSA 1 mg / ml; MgSO4 3 mM, DMSO 0.5% and Triton X-100 0.5%.

[0064] According to the number of reactions N, mix equal volumes of amplification buffer and amplification reagent, shake and mix, centrifuge briefly, and obtain amplification mixture for use. Add 45 μl of amplification mixture to 30 μl of pre-amplification reaction product in the previous step and run the following program.

[0065] Table 3

[0066]

[0067] 3. Quality inspection of amplified products, library construction, and computerization

[0068] 1) Magnetic bead recovery of amplified products (CW2508), product qPCR reaction

[0069] Take 5ul PCR product + 1ul loading buffer for electrophoresis and run on 1% agarose gel.

[0070] Take 60ul+120ul magnetic beads (CW2508) for purification, elute with 60ul ddH2O, and measure the DNA concentration with Qubit.

[0071] The purified DNA of the amplified product was subjected to fluorescence quantitative experiment.

[0072] The specific primers used for amplification product detection are:

[0073] Table 4

[0074]

[0075] Reaction system:

[0076] Table 5

[0077]

[0078] Reaction procedure:

[0079] Table 6

[0080]

[0081]

[0082] 2) Amplification product interruption and library construction (CW3025)

[0083] 4. Comparative analysis of offline data

[0084] At the same time, a domestic kit and Takara reagent were used for comparative testing. The electrophoresis comparison results are as follows Figure 1 As shown, the domestic kit used in the comparison cannot amplify single cells. The method of the present invention can effectively amplify culture fluid containing 1-5 cells, and is significantly better than Takara reagent (Cat. No.: R300671, amplification is performed under the conditions described in the kit manual), and the amplification product qPCR detection is shown in Table 7.

[0085] Table 7

[0086]

[0087] Among them, for the amplification of one cell, only three pairs of primers were used to amplify the target nucleic acid using the Takara amplification reagent, and the average Ct value could not be calculated.

[0088] All eight primer pairs were able to amplify effectively, and the average Ct value of amplification and the SD value of the Ct values ​​of the eight primer pairs were better than those of Takara reagent.

[0089] The amplified product was fragmented by enzyme digestion and library preparation (CW3025 DNA library rapid preparation kit containing fragmentation enzyme (Illumina & MGI)), and the copy number variation analysis of the data off the machine was shown in the attached Figure 2 , the amplified product library can effectively perform copy number variation analysis.

[0090] Example 2. Comparison of whole genome amplification in simulated cell culture medium containing trace amounts of genome

[0091] 1. Preparation of simulated culture medium

[0092] The genome quantification kit of Jiangsu Kangwei Century Biotechnology Co., Ltd., product number CW2683, was used to accurately quantify the human genome. The quantified template was diluted to the required level with DMEM and G-2 culture medium respectively.

[0093] Steps 2-4 are the same as in Example 1

[0094] At the same time, a domestic kit and Takara reagent were used for comparative testing. The electrophoresis comparison results are as follows Figure 3 As shown in the figure, the domestic kit used in the comparison cannot amplify 15pg sample. The method of the present invention can effectively amplify culture fluid containing trace gDNA (7.5pg-30pg), which is significantly better than Takara reagent. The amplification product qPCR detection is shown in Table 8.

[0095] Table 8

[0096]

[0097] All eight primer pairs were able to amplify effectively, and the average Ct value of amplification and the SD value of the Ct values ​​of the eight primer pairs were better than those of Takara reagent.

[0098] The amplified product was fragmented by enzyme digestion and library preparation (CW3025 DNA library rapid preparation kit containing fragmentation enzyme (Illumina & MGI)), and the copy number variation analysis of the data off the machine was shown in Figure 4 , the amplified product library can effectively perform copy number variation analysis.

[0099] Comparative Example 1:

[0100] When whole genome amplification of trace genes is performed with cell culture fluid samples, the presence of amplification inhibitors in the samples results in poor amplification effects and inability to amplify extremely trace target genes. The present invention significantly reduces the detection limit of whole genome amplification of trace genes by improving the amplification procedure and the amplification buffer. During the research process, different amplification procedures and amplification buffers were optimized and improved, and finally anti-inhibition amplification was achieved, greatly improving the detection sensitivity.

[0101] This comparative example demonstrates the amplification effect of the amplification procedure before improvement.

[0102] Using the same reaction system as in Example 1, the pre-amplification runs the following program:

[0103] Table 9

[0104]

[0105] Amplification reactions were run as follows.

[0106] Table 10

[0107]

[0108] The purified DNA of the amplified product was subjected to a fluorescence quantitative experiment, and the specific steps were the same as those in Example 1, and the results were as follows:

[0109] Table 11

[0110] serial number 1 cell amplification product / Ct 5 cell amplification products / Ct Primer 1 / / Primer 2 / 29.48 Primer 3 / / Primer 4 32.51 30.05 Primer 5 / / Primer 6 / 31.27 Primer 7 / 29.86

[0111]

[0112] Note: “ / ” indicates that effective amplification cannot be performed.

[0113] Among them, in the amplification of a single cell, only primer pair 4 and primer pair 8 among the 8 primer pairs were able to perform effective amplification. In the amplification of 5 cells, 5 primer pairs among the 8 primer pairs were able to perform effective amplification.

[0114] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for whole genome amplification, characterized in that: The method comprises the steps of: S1. Provide a sample containing a trace amount of genomic DNA as a template; S2. Pre-amplify the template in the sample using a degenerate oligonucleotide primer to obtain a pre-amplification product, wherein the pre-amplification reaction procedure includes: Denature at 95°C-98°C for 1-5 min; pre-denature at 95°C-98°C for 10-30 s, incubate at the first temperature for 40-60 s, incubate at the second temperature for 30-50 s, incubate at the third temperature for 20-40 s, incubate at the fourth temperature for 30-50 s, incubate at the fifth temperature for 30-50 s, for a total of 10-15 cycles; Wherein, the first temperature is 10°C-20°C, the second temperature is 5°C-15°C higher than the first temperature, the third temperature is 5°C-15°C higher than the second temperature, the fourth temperature is 20°C-40°C higher than the third temperature, the fifth temperature is 5°C-15°C higher than the fourth temperature, and the fifth temperature is not higher than 80°C; S3. Amplify the pre-amplification product using degenerate oligonucleotide primers.

2. The method according to claim 1, characterized in that The sample in step S1 contains cell culture medium or embryo culture medium.

3. The method according to claim 2, characterized in that The cell culture medium includes commercial cell culture medium such as 1640 and DMEM; the embryo culture medium includes embryo culture medium such as Vitrolife G-2 and SAGE ART.

4. The method according to claim 1, characterized in that The sample contains genomic DNA from 1 to 10 cells; or, the sample contains 5 to 30 pg / ul of gDNA.

5. The method according to claim 1, characterized in that In the step S1, a cell culture medium containing 1-10 cells is treated with a cell lysing enzyme to obtain a template containing a trace amount of genomic DNA.

6. The method according to claim 1, characterized in that The cell lytic enzyme comprises one or more of proteinase K, QIAGEN Protease, pepsin, trypsin, lysozyme, etc.; preferably proteinase K.

7. The method according to claim 1, characterized in that The pre-amplification reaction system for pre-amplification in step S2 comprises a pre-amplification reagent and a pre-amplification buffer, wherein the pre-amplification reagent comprises one or more components selected from the group consisting of a DNA polymerase, a degenerate oligonucleotide primer, and a dNTP; and the pre-amplification buffer comprises Tris-Cl, KCl, BSA, MgSO4, DMSO, and Triton X-100.

8. The method according to claim 1, characterized in that The reaction procedure for amplification in step S3 includes: Denature at 95℃-98℃ for 1-5min; pre-denaturation at 95℃-98℃ for 10-30s, incubate at 60℃-70℃ for 30-120s, incubate at 70℃-80℃ for 30-120s, for a total of 10-20 cycles.

9. The method according to claim 1, characterized in that The amplification reaction system for amplification in step S3 comprises an amplification reagent and an amplification buffer, wherein the amplification reagent comprises one or more components selected from the group consisting of: DNA polymerase, degenerate oligonucleotide primers and dNTPs; and the amplification buffer comprises Tris-Cl, KCl, BSA, MgSO4, DMSO, and Triton X-100.

10. The method according to claim 1, characterized in that The DNA polymerase is selected from the group consisting of Phi29 DNA polymerase, Bst DNA polymerase, Vent polymerase, Deep Vent polymerase, Klenow Fragment DNA polymerase I, super-fidelity DNA polymerase, Taq polymerase, E. coli DNA polymerase, Long Amp Taq DNA polymerase and One Taq DNA polymerase; preferably Phi29 DNA polymerase.

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