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Extracting method for metagenome DNA (Deoxyribonucleic Acid)

An extraction method and metagenomics technology, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problem that host cells cannot be removed

Inactive Publication Date: 2016-11-23
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only remove the insect tissue DNA wrapped outside the gut, and cannot remove the host cells present in the gut

Method used

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  • Extracting method for metagenome DNA (Deoxyribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Metagenomic DNA extraction of stool samples

[0026] Take two clean 5ml centrifuge tubes, weigh 1g of two different fecal samples No. 1 and No. 2 into the centrifuge tubes, add 1.6ml of Buffer SLX Mlus to the two tubes, shake and mix evenly. Weigh 500 mg of glassbeads into four 2 mL centrifuge tubes numbered A, B, C, and D, respectively, and then add 5 μL of proteinase K at a concentration of 10 mg / mL to the centrifuge tubes numbered A and B, respectively. Then add 1 mL of No. 1 stool sample to centrifuge tubes numbered A and C, respectively, and add 1 mL of No. 2 stool sample to centrifuge tubes numbered B and D respectively. The components in the centrifuge tubes numbered A, B, C, and D (hereinafter referred to as tubes A, B, C and D) are shown in Table 1:

[0027]

A

B

C

D

stool sample

1

2

1

2

buffer

1.6mL

1.6mL

1.6mL

1.6mL

Proteinase K

5μL

5μL

0

0

glass beads

500...

Embodiment 2

[0039] Example 2 Metagenomic DNA extraction of blood samples

[0040] Two different blood samples were taken to extract metagenomic DNA according to the method of Example 1. The blood samples in tubes A and B were added with 5 μL proteinase K at a concentration of 15 mg / mL, heated at 60 °C for 20 min to lyse the host cells by proteinase K, and then heated at 80 °C for 20 min to inactivate proteinase K, and then followed Extract metagenomic DNA according to the instructions of the kit. The blood samples in tubes C and D were directly extracted for metagenomic DNA according to the operating steps of the kit instructions.

[0041] The above metagenomic DNA from blood samples was detected by 2% agarose gel electrophoresis. figure 1 similar. Then, library construction and sequencing analysis were performed. The results are shown in Table 4. The content of host DNA in metagenomic DNA without proteinase K heat treatment was about 6 times that after proteinase K heat treatment.

...

Embodiment 3

[0044] Example 3 Metagenomic DNA extraction of tissue samples

[0045] Two different human intestinal tissue samples were taken, and metagenomic DNA was extracted according to the method of Example 1. The intestinal tissue samples in tubes A and B were added with 5 μL of proteinase K at a concentration of 20 mg / mL, heated at 55 °C for 30 minutes to lyse the host cells by proteinase K, and then heated at 90 °C for 15 minutes to inactivate proteinase K, then Then, extract metagenomic DNA according to the operating steps of the kit instructions. The intestinal tissue samples in tubes C and D were directly extracted from metagenomic DNA according to the instructions of the kit.

[0046] The above metagenomic DNA from intestinal tissue samples was detected by 2% agarose gel electrophoresis. figure 1 similar. Then, library construction and sequencing analysis were performed. The results are shown in Table 5. The content of host DNA in metagenomic DNA without proteinase K heat tre...

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Abstract

The invention provides a method for extracting metagenomic DNA. The extraction method comprises the following steps: firstly, adding a buffer solution and proteinase K to a sample to be tested to obtain a mixed solution, and heating the mixed solution so that the buffer solution and proteinase K are cracked. The host cells in the test sample are further heated to inactivate proteinase K, and then the microbes are broken by beating, and then the DNA is extracted to obtain the metagenomic DNA. In the invention, the lysate is firstly added to suspend the host cell, and then proteinase K is added to degrade the protein of the host cell and free the DNA. Due to the absence of lysozyme and external force, bacterial microorganisms can maintain a complete form. In the process of beating to break the microbial wall, on the one hand, the bacteria and microorganisms are broken, and on the other hand, the host DNA is broken into small fragments or even degraded. Finally, the metagenomic DNA is extracted according to the conventional method, which can reduce the background interference of the host DNA .

Description

technical field [0001] The invention relates to the field of metagenomic detection, in particular to a method for extracting metagenomic DNA. Background technique [0002] Meta-genome, also known as metagenome, refers to the sum total of all microbial genetic material in a specific environment. It directly extracts the DNA of all microorganisms from environmental samples, constructs a metagenomic library, and uses the research strategy of genomics to study the genetic composition and community functions of all microorganisms contained in environmental samples. [0003] The species and abundance of microorganisms in animal intestines, body fluids and other tissues greatly affect the living state of the host itself. The human gut, for example, is home to 10 trillion bacteria that affect weight and digestion, fight infection and reduce the risk of autoimmune diseases, and control the body's response to cancer treatments. Once the intestinal flora is imbalanced, a series of di...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 李静陈昌岳张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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