Method for acquiring high-quality soil microbial genome DNA with pretreatment means

A soil microorganism and pretreatment technology, applied in the field of molecular biology, can solve the problem of sacrificing diversity and achieve the effect of wide range of objects, obvious removal effect and high quality

Inactive Publication Date: 2011-06-01
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Methods of diluting template genomic DNA can sometimes solve this problem, but at the expense of diversity

Method used

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  • Method for acquiring high-quality soil microbial genome DNA with pretreatment means
  • Method for acquiring high-quality soil microbial genome DNA with pretreatment means
  • Method for acquiring high-quality soil microbial genome DNA with pretreatment means

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The Taoyuan Station of the Institute of Subtropical Agroecology, Chinese Academy of Sciences was selected as the research site, and paddy field soil samples of 5-15 cm were collected as the research object. The physical and chemical properties of the soil were measured (pH 4.96, total organic carbon content 2.37%). Weigh 5g of soil sample, put it in a 50ml centrifuge tube, add 5ml of calcium carbonate suspension (1M), mix by inverting several times, place it at room temperature for 1h, and mix gently by inverting at intervals of 10min. After being treated with calcium carbonate for 1 h, centrifuge at 10000×g for 10 min at room temperature, and take 0.5 g of the precipitate The kit extracts soil microbial genomic DNA, and the extraction method refers to the protocol provided by the manufacturer along with the kit. For the control experiment, 0.5g soil sample without any pretreatment was directly weighed, using The kit extracts soil microbial genomic DNA. figure 1 It ...

Embodiment 2

[0020] The Qiyang Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences was selected as the research site, and dry land soil samples of 5-15 cm were collected as the research object. The physical and chemical properties of the soil were measured (pH 5.63, total organic carbon content 0.8%). Operations such as extraction of soil microbial genomic DNA, PCR amplification, and DGGE electrophoresis are the same as in Example 1. The DNA extracted from the soil sample pretreated with calcium carbonate was used as a template, and the Shannon index of the DGGE spectrum of the product after PCR amplification was 3.2. Correspondingly, the Shannon index of the soil samples without pretreatment is less than 3. Calculation results of Shannon index showed that using DNA extracted from calcium carbonate pretreated soil samples as templates, PCR amplification products had more diversity.

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Abstract

The invention relates to a method for acquiring high-quality soil microbial genome DNA through re-extraction by pretreating a soil sample. In the invention, a solution of calcium carbonate and the like is used for pretreating a soil sample to be subjected to genome DNA extraction, the extracted DNA has clear appearance color, and the DNA with OD260/OD280 of 1.81 has high purity. The extracted DNA can be used for PCR (Polymerase Chain Reaction) amplification directly, and a DGGE (Denaturing Gradient Gel Electrophoresis) map indicates that an amplified product has high concentration and sharp banding patterns. Shannon index indicates that the amplified product has great diversity and can effectively reflect the microorganism diversity of a soil microbial community. The pretreatment technology provided by the invention is simple and feasible, has the advantages of low cost, obvious effect on removing humic acid substances and high quality of extracted genome DNA, and is suitable for the extensive research field of molecular ecology.

Description

Technical field: [0001] The invention relates to the technical field of molecular biology, in particular to certain processing of soil samples to obtain high-quality soil microbial genome DNA. Background technique: [0002] Molecular ecology techniques that do not depend on cultivation have gradually become a powerful means to analyze and understand the diversity of soil microorganisms and their physiological characteristics under in situ conditions. At present, molecular ecology has been involved in many research fields, providing a lot of valuable information for the study of environmental microbial community diversity and ecophysiological functions under in situ conditions. The foundations of molecular ecology depend on high-quality DNA templates. There are many methods for extracting soil microbial genomic DNA at present, and commercialized kit methods (such as etc.) and methods in some literatures (such as Zhou’s method, etc.) can basically meet the needs of extracti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04
Inventor 庄国强吕迪马安周
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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