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Efficient extraction method of dendrobium officinale genome DNA

Dendrobium officinale and extraction method technology, applied in the field of molecular biology, can solve problems such as difficult to dissolve, difficult to identify, and insufficient removal of polysaccharides, and achieve the effect of easy extraction process, easy extraction and removal, and beneficial for building a database on the machine

Inactive Publication Date: 2020-07-24
WUHAN FRASERGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classical plant genome extraction method is mainly the CTAB method, and the Chinese herbal medicine plant containing polysaccharides and polyphenols and other secondary metabolites similar to Dendrobium, in the process of DNA extraction, polyphenols are very easy to oxidize, after oxidation The product of the polysaccharide is irreversibly combined with the DNA molecule, and at the same time, the polysaccharide forms a sticky gel-like complex with the DNA, which makes the extracted DNA sample not only brown and difficult to dissolve, but also difficult to be recognized by restriction enzymes and TaqDNA polymerase , resulting in the failure of PCR amplification and enzyme digestion, so that the molecular biology research of Dendrobium candidum is progressing slowly
At present, many people have carried out research on the DNA extraction of Dendrobium, but most of them are for small amount of extraction, and the removal of polysaccharides is not thorough enough.

Method used

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  • Efficient extraction method of dendrobium officinale genome DNA
  • Efficient extraction method of dendrobium officinale genome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for efficiently extracting genomic DNA of Dendrobium officinale, the method comprising the steps of:

[0031] (1) Take an appropriate amount of Dendrobium officinale leaves and put them into a mortar, grind them into powder with liquid nitrogen, and put 1.0 g of the powder into a centrifuge tube after grinding.

[0032] (2) Add 8 mL of extraction buffer to the powder, and bathe in water at 65°C for 25 minutes to obtain mixture a; the formula of the extraction buffer is: 100 mM Tris-HCl (pH8.0), 20 mM EDTA, 1.4 M NaCl, 2% (W / V ) CATB, 20% (W / V) PVP, 0.4% (V / V) β-mercaptoethanol.

[0033] (3) Add pectinase to mixed solution a so that the final concentration in mixed solution a is 40 U / mg, and incubate at 37° C. for 10-20 minutes to obtain mixed solution b.

[0034] (4) Add an equal volume of a mixed solution of phenol, chloroform and isoamyl alcohol to the mixed solution b, mix thoroughly and centrifuge at 4°C and 6000 rpm for 10 minutes to obtain supernatant a;...

Embodiment 2

[0040] A method for efficiently extracting genomic DNA of Dendrobium officinale, the method comprising the steps of:

[0041] (1) Put an appropriate amount of Dendrobium leaves into a mortar, grind them into powder with liquid nitrogen, and put 1.0 g of the powder into a centrifuge tube after grinding.

[0042] (2) Add 6 mL of extraction buffer to the powder, and bathe in 70°C for 20 minutes to obtain mixture a; the formula of extraction buffer is: 100 mM Tris-HCl (pH8.0), 20 mM EDTA, 1.4M NaCl, 2% (W / V ) CATB, 20% (W / V) PVP, 0.4% (V / V) β-mercaptoethanol.

[0043] (3) Add pectinase to mixed solution a (the final concentration of pectinase in mixed solution a is 30 U / mg), and incubate at 35° C. for 10-20 minutes to obtain mixed solution b.

[0044] (4) Add an equal volume of mixed solution of phenol, chloroform and isoamyl alcohol to the mixed solution b, and after thorough mixing, centrifuge at 25°C and 9000rpm for 8 minutes to obtain supernatant a; the volume ratio of phenol...

Embodiment 3

[0050] A method for efficiently extracting genomic DNA of Dendrobium officinale, the method comprising the steps of:

[0051] (1) Put an appropriate amount of Dendrobium leaves into a mortar, grind them into powder with liquid nitrogen, and put 1.0 g of the powder into a centrifuge tube after grinding.

[0052] (2) Add 10mL of extraction buffer to the powder, and bathe in water at 60°C for 30min to obtain mixture a; the formula of extraction buffer is: 100mM Tris-HCl (pH8.0), 20mM EDTA, 1.4MNaCl, 2% (W / V ) CATB, 20% (W / V) PVP, 0.4% (V / V) β-mercaptoethanol.

[0053] (3) Add pectinase to mixed solution a (the final concentration of pectinase in mixed solution a is 50 U / mg), and incubate at 40° C. for 10-20 minutes to obtain mixed solution b.

[0054] (4) Add an equal volume of a mixed solution of phenol, chloroform and isoamyl alcohol to the mixed solution b, mix well and then centrifuge at 37°C and 12000rpm for 5min to obtain supernatant a; the volume ratio of phenol, chlorofo...

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Abstract

The invention relates to the technical field of molecular biology, in particular to an efficient extraction method of dendrobium officinale genome DNA. According to the method, the formula of an extraction buffer solution in a classical CTAB method is optimized, one-step pectinase cracking is added, and the extraction process is optimized. Compared with a classical CTAB method, the DNA extractionmethod has the advantages that spontaneous oxidation of phenol can be well inhibited and DNA browning reaction caused by polyphenol oxide can be prevented in the extraction buffer solution treatment process, polysaccharide is promoted to be combined with high-concentration CTAB under the high-salt condition, and therefore extraction and removal are facilitated; insoluble substances in dendrobium officinale are further dissolved in the pectinase treatment process, so that the extracted DNA is not viscous any more, and the subsequent DNA sample library building and sequencing processes are remarkably simplified. According to the extraction method disclosed by the invention, the DNA extraction efficiency and the DNA purity are obviously improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for efficiently extracting genomic DNA of Dendrobium officinale. Background technique [0002] Dendrobium officinale Kimura et Migo is a perennial herbaceous epiphytic plant of the genus Dendrobium officinale. It is the original plant of the famous and precious Chinese medicine Xifengdou. The key medicine, in the medical classic "Tao Cang" of the Tang Dynasty, is known as "the first of the nine immortal grasses in China". In 1987, it was included in the list of endangered and rare plants under national key protection. Dendrobium officinale is rich in polysaccharides and other active ingredients. Modern medical research has shown that it has good curative effects on throat diseases, cardiovascular and cerebrovascular diseases, diabetes, cataracts, etc. It also has anti-tumor, anti-aging, enhancing human immunity and anti-inflammatory effects. [0003] Genomic D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 吴冬晴
Owner WUHAN FRASERGEN CO LTD
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