Method for extracting macro fungus genome DNA

A genome and fungal technology, applied in the field of extracting large fungal genome DNA, can solve the problems of low DNA yield, unpurified treatment, cumbersome steps, etc., and achieve the effect of high purity, high yield and clear interface

Inactive Publication Date: 2018-05-01
SHANGHAI ACAD OF AGRI SCI
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Problems solved by technology

Lu Zuozhou et al. (Edible Fungi Genetic Engineering Experimental Technology [J]. Chinese Edible Fungi, 1992, 11 (1): 13) reported a method for DNA extraction from edible fungi, but this method needs to prepare protoplasts, which is time-consuming and expensive to extract. The yield of DNA is low; Borroso G et al. (A miniprep method for RFLP analysis and dsRNAS detection perfected in the cutivated fungus Agrocybe aegerita. Science and Cultivation of Edible Fungi, 1995, 87-94) used the CTAB method to extract genomic DNA from mushrooms, but the steps are cumbersome , low DNA yield; Yang Tongwen et al. (CTAB combined with DNA gel recovery kit to extract DNA from edible fungi [J]. Biotechnology, 2009, 19(1): 32-34) established a CTAB combined with DNA gel kit to extract edible mushroom DNA. The method of bacterial DNA does not use phenol imitation extraction and DNA drying, redissolution and other operations, but the cost of this method is relatively high; Sun Lifu et al. , 28(2):299-302) extracted fungal DNA by grinding with a small manual electric drill with a sterile plastic drill bit, but this method was time-consuming
Chinese patents ZL2010101628359 and ZL2010101628452 provide methods for extracting genomic DNA from edible fungi. Although these two methods can complete DNA extraction in a relatively short period of time, they do not carry out subsequent purification treatment on the extracted DNA. For some DNA purity requirements Higher experiments can adversely affect

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  • Method for extracting macro fungus genome DNA
  • Method for extracting macro fungus genome DNA
  • Method for extracting macro fungus genome DNA

Examples

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Embodiment 1

[0045] Utilize the inventive method to extract the genomic DNA of 6 different macrofungi samples (fruiting bodies and hyphae of straw mushroom, shiitake mushroom and Hericium erinaceus) respectively, and the extraction steps are as follows:

[0046] 1. Take 100 mg of fungal fruiting bodies or 20 mg of fungal hyphae, grind them into powder in liquid nitrogen, add 600 μl of preheated reagent I, mix well, warm bath at 65°C for 30 minutes, and centrifuge at 12,000 rpm for 10 minutes.

[0047] 2. Transfer the supernatant to a sterilized 1.5ml centrifuge tube, add an equal volume of reagent II, mix well for 2 minutes, and centrifuge at 12,000rpm for 10 minutes.

[0048] 3. Draw the supernatant into a sterilized 1.5ml centrifuge tube, add isopropanol equal to the volume of the supernatant, mix well, centrifuge at 12000rpm for 10min, discard the supernatant, keep the precipitate, add 60μl of reagent III, use the specification model 200μl sterilized pipette tip to thoroughly mix the pr...

Embodiment 2

[0067] In this example, the content of each component in reagent I is: Tris-HCl 250mM, NaCl 250mM, EDTA 25mM, SDS cell lysate 0.45wt.%, vinylpolypyrrolidone 0.5wt.%, pH=7.8; each component in reagent II The content is: phenol, chloroform and isoamyl alcohol volume ratio (V / V=25:24:1); reagent III is: 1×TE buffer; the content of each component of reagent IV is: guanidine isothiocyanate 8M, Tris -HCl 40mM, EDTA 45mM, Triton X-100 25mM; the content of each component of reagent V is: guanidine isothiocyanate 8M, Tris-HCl 40mM; the content of each component of reagent VI is: Tris-HCl 40mM, NaCl 250mM, ethanol 73vol%.

[0068] Other operating steps are the same as in Example 1, and the DNA concentration and purity results of the extracted water body samples are shown in Table 2.

[0069] Table 2

[0070]

[0071] It can be seen from Table 2 that the concentration of the obtained DNA is about 20-50 ng / μl, the ratio of OD260 / OD280 is between 1.8-2.0, and the purity and yield are ...

Embodiment 3

[0074] In this example, the content of each component in reagent I is: Tris-HCl 300mM, NaCl 300mM, EDTA 30mM, SDS cell lysate 0.5wt.%, vinylpolypyrrolidone 0.6wt.%, pH=8.0; each component in reagent II The content is: phenol, chloroform and isoamyl alcohol volume ratio (V / V=25:24:1); reagent III is: sterilized distilled water; the content of each component of reagent IV is: guanidine isothiocyanate 9M, Tris-HCl 50mM, EDTA 50mM, Triton X-100 30mM; the content of each component of reagent V is: guanidine isothiocyanate 9M, Tris-HCl 50mM; the content of each component of reagent VI is: Tris-HCl 50mM, NaCl 300mM, ethanol 75vol% .

[0075] Other operating steps are the same as in Example 1, and the results of DNA concentration and purity of the extracted water samples are shown in Table 3.

[0076] table 3

[0077]

[0078] It can be seen from Table 3 that the concentration of the obtained DNA is about 20-50 ng / μl, the ratio of OD260 / OD280 is between 1.8-2.0, and the purity an...

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Abstract

The invention discloses a method for extracting macro fungus genome DNA. A macro fungus fruiting body or hypha cell structure is broken via a physical method and a chemical method; protein is removedby virtue of phenol, chloroform and isoamylol; then, genome DNA is precipitated by virtue of isopropanol; sterilized distilled water or a buffer solution is added to dissolve the genome DNA; the genome DNA is adsorbed and collected by virtue of a nucleic acid adsorption column; and purifying is conducted by virtue of guanidinium isothiocyanate and ethanol, so that the genome DNA is obtained. The obtained genome DNA is relatively good in integrity; through dyeing after agarose gel electrophoresis, a DNA strip is clear and single, without tailing or a complex zone; the obtained genome DNA is high in purity and high in yield, an OD260 to OD280 ratio ranges from 1.8 to 2.0, and the concentration of the genome DNA is 20-50ng/ul; and the method is simple to operate and low in cost, and is applicable to molecular biology studies of various macro fungus fruiting bodies or hyphae.

Description

technical field [0001] The invention belongs to the field of DNA extraction, and in particular relates to a method for extracting large fungus genome DNA. Background technique [0002] Macrofungi refers to a class of fungi that can form fleshy or gelatinous fruiting bodies, which are divided into edible, medicinal, and poisonous fungi. Macrofungi are an important group of fungi, and many species have high nutritional and medicinal value, and are currently the most promising category of fungi. In recent years, with the rapid development of modern biotechnology, molecular biology techniques such as molecular markers and genetic engineering have been widely used in the study of macrofungi. The extraction, separation and purification of genomic DNA of macrofungi is one of the key technologies. Large fungi are rich in proteins and polysaccharides, and the presence of proteins and polysaccharides will affect DNA digestion, PCR reactions and Southern hybridization experiments. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12N15/101
Inventor 李鹏唐雪明潘爱虎蒋玮王金斌武国干吕贝贝吴潇贾军伟王荣谈白蓝刘华王慧叶吉妮
Owner SHANGHAI ACAD OF AGRI SCI
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