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Extraction method of microbe DNA for high-throughput sequencing in sample

An extraction method and high-throughput technology, which is applied in the fields of high-throughput gene sequencing and DNA extraction, can solve problems such as difficulties, low cell lysis rate, and inaccessibility, and achieve fast extraction speed, low degradation degree, and good versatility Effect

Inactive Publication Date: 2015-12-23
BIOMARKER TECH
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  • Abstract
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  • Claims
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Problems solved by technology

In addition, there are large differences between different soil samples, so that different types of soil will affect the extraction effect of microbial DNA, resulting in the problem of low yield of DNA extraction
[0004] In the existing technology, the extraction rate of soil microbial DNA is low, and it is even more difficult to efficiently extract DNA from environmental samples, which cannot meet the requirements of high-throughput sequencing library preparation for microbial diversity sample DNA: 1.8 ≤OD260:280≤2.0, DNA intact without degradation or slight degradation
[0005] Based on the above points, it is necessary to develop a convenient, fast, and practical method for soil microbial DNA extraction suitable for high-throughput sequencing, to solve the problem of the presence of PCR inhibitors such as humic substances in DNA samples obtained by conventional methods, as well as low cell lysis rates and DNA The problem of low yield
Aiming at the difficulties and characteristics of microbial DNA extraction in soil, some domestic and foreign biotechnology companies have developed commercial kits for extracting microbial DNA in soil, but the effect of extracting environmental microbial DNA is not ideal

Method used

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  • Extraction method of microbe DNA for high-throughput sequencing in sample
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  • Extraction method of microbe DNA for high-throughput sequencing in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 The present invention is used for the preparation of the reagent of extracting microbial DNA

[0037] The reagent consumables for extracting microbial DNA according to the present invention include:

[0038] Lysis buffer contains: 100mM Tris-HCl, 100mM EDTA, 100mM Na 3 PO 4 , 1.5M NaCl, and 0.1% CTAB. Specific configuration method: weigh 0.82gNa respectively 3 PO 4 and 0.5gCTAB, add appropriate amount of distilled water to dissolve respectively, then add 5mL of 1M Tris-HCl, 10mL of 0.5M EDTA, 15mL of 5M NaCl, and then make the volume to 50mL.

[0039] Aluminum ammonium sulfate solution: 50-200mM Al 2 (SO 4 ) 3 and 50-200mM (NH 4 ) 2 SO 4 , the two are mixed according to the volume ratio of 1:1. The aluminum ammonium sulfate solution in the embodiment of the present invention selects the Al of 120mM for use 2 (SO 4 ) 3 and 120mM (NH 4 ) 2 SO 4 It is obtained by mixing in a volume ratio of 1:1.

[0040] Binding solution: 5M guanidine hydroch...

Embodiment 2

[0044] Example 2 Microbial DNA Extraction and Effect Verification from Soil

[0045] The reagents and consumables described in Example 1 were used for extraction.

[0046] (1) the method for extracting microbial DNA in the soil of the present invention

[0047] 1. Weigh about 500mg of soil sample into a 2mL centrifuge tube, add 1.25mL of lysis buffer, add 25uL of β-mercaptoethanol at the same time, and vortex for 2 minutes. Then 30 μL of lysozyme (15 mg / mL) was added and placed on a shaker at 37° C. for 15 min.

[0048] 2. Add 250 μL of 10% SDS and 20 μL of proteinase K (20 mg / mL) solution, and immediately vortex to mix thoroughly. Water bath at 65°C for 10 minutes, and mix by inverting every 2 minutes during this period.

[0049] 3. Centrifuge at 10000g for 2min at room temperature, and transfer the supernatant to a clean 1.5mL centrifuge tube.

[0050] 4. Add 1 / 3 volume of 5M NaCl solution to the supernatant, vortex and mix for 5 seconds, and place in an ice bath at 4°C ...

Embodiment 3

[0061] Example 3 Microbial DNA Extraction and Effect Verification from Feces

[0062] The reagents and consumables described in Example 1 were used for extraction.

[0063] The method for extracting microbial DNA in feces of the present invention:

[0064] 1. Weigh about 500mg of cryopreserved cow manure sample into a 2mL centrifuge tube, add 25uL β-mercaptoethanol and 1.25mL lysis buffer, and vortex for 2 minutes. Then 30 μL of lysozyme (15 mg / mL) was added and placed on a shaker at 37° C. for 15 min.

[0065] 2. Add 250 μL of 10% SDS and 20 μL of proteinase K (20 mg / mL) solution, and immediately vortex to mix thoroughly. Water bath at 65°C for 10 minutes, and mix by inverting every 2 minutes during this period.

[0066] 3. Centrifuge at 10000g for 2min at room temperature, and transfer the supernatant to a clean 1.5mL centrifuge tube.

[0067] 4. Add 1 / 3 volume of 5M NaCl solution to the supernatant, vortex and mix for 5 seconds, and place in an ice bath at 4°C for 8 min...

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Abstract

The invention provides an extraction method of a microbe DNA for high-throughput sequencing in a sample and belongs to the field of a genome DNA extraction technology. According to the invention, DNA of a microbe in a sample is extracted by a SDS method; an inhibitor is removed after precipitation by the use of a sodium chloride solution and a aluminum ammonium sulfate solution, and then a supernatant is taken; and separation and purification are carried out by a silica gel film-column to obtain soil microbe genome DNA for high-throughput sequencing. The aluminum ammonium sulfate solution is prepared from 50-200 mM of Al2(SO4)3 and 50-200 mM of (NH4)2SO4 according to volume ratio of 1:1. The sample's microbe genome DNA extracted by the above method has high purity, good universality and fast extraction speed, can be directly used in a downstream high-throughput sequencing experiment and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of gene high-throughput sequencing and DNA extraction, in particular to a method for extracting microbial DNA from samples suitable for high-throughput sequencing. Background technique [0002] Microorganisms refer to the general term of a group of tiny organisms that are invisible or unclear to the naked eye, and are a large group of organisms including bacteria, viruses, fungi, and some small protozoa, microscopic algae, etc. They are small in size, simple in structure, and their application fields are expanding day by day. They are closely related to human daily life and health, and their industrial applications are also becoming more and more extensive. The microbial resources in the soil are rich, and the soil is an important part of the terrestrial ecosystem. It is very sensitive to the external environment, has poor stress resistance, and is easily affected by various adverse external environments. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06
Inventor 郑洪坤刘慧刘少卿何超
Owner BIOMARKER TECH
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