Extraction and purification method of DNAs of fagaceae plant sample

A purification method, the technology of Fagaceae, applied in the field of plant DNA extraction and purification, can solve the problems of high price, unstable chemical properties of DNA, protein polyphenol pollution, etc., and achieve the effect of preventing polyphenol pollution

Inactive Publication Date: 2019-01-04
SHANGHAI INST OF TECH
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AI Technical Summary

Problems solved by technology

However, when these general extraction methods are used to extract plants with a high content of polysaccharides and polyphenols, they often cannot obtain high-purity genomic DNA
The obtained total DNA is brown in color and is wrapped by jelly-like polysaccharide substances. Agarose electrophoresis shows that the DNA has tailing phenomenon, the sample well is bright, and the pollution of proteins, polysaccharides and polyphenols is serious, so it is difficult to carry out downstream experimental analysis. Commercially available The kit is expensive, and the yield of extracted DNA is not high, and the cost performance is poor
[0006] In addition, due to the long-term preservation of plant specimens, the chemical properties of DNA are unstable, and they are easy to spontaneously degrade through hydrolysis or oxidation. Therefore, the older the specimen, the more difficult it is to extract DNA.
In addition, the specimens are disturbed by physical and chemical factors such as high temperature and insecticides and fungicides during the production and storage process, resulting in a large amount of degradation and low content of DNA contained in the specimens. During the extraction process, it is easy to cause serious browning and pigment pollution due to oxidation. Seriously, coupled with the low yield, more samples are required than general materials, but some specimen materials are very limited and precious. It is necessary to effectively extract high-purity genomic DNA from these precious materials for molecular biology experiments such as next-generation sequencing. seems particularly urgent
[0007] At present, there are many reports and many patents on extracting DNA from animal specimens, but it is difficult to extract DNA from plant specimens, and there is no mature general method.

Method used

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  • Extraction and purification method of DNAs of fagaceae plant sample
  • Extraction and purification method of DNAs of fagaceae plant sample
  • Extraction and purification method of DNAs of fagaceae plant sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The DNA extraction process of the silica gel dry leaves and fresh leaves of Quercus thorny leaf, Quercus japonica, Qinggang, Fructus japonica, Quercus chinensis, and Quercus trigonum provided by the present embodiment is as follows:

[0075] (1) Both fresh leaves and silica gel-dried leaves were cut into 2×2cm squares, ground into powder in liquid nitrogen, and used for DNA extraction;

[0076] (2) Add 1ml of special rinse solution (2% PVP, 1% L-Ascorbic Acid, 3% Triton-X 100, 4% β-mercatoeyhanol) to the ground powder, shake, centrifuge at 10000rpm for 1min, and discard the supernatant ( If the supernatant is viscous, repeat this step until the supernatant is colorless and has a clear boundary with the precipitate);

[0077] (3) Add 800 μL of 2% CTAB extract (2% CTAB (m / v), 1.4M NaCl, 100mM Tris-HCl pH 8.0, 20mM EDTA pH 8.0, 10% (w / v) N- Lauroylsarcosine Sodium salt, 1.4% β-mercatoeyhanol, 50mg RNase A), placed in a water bath at 60°C for 40 minutes, and gently mixed u...

Embodiment 2

[0083] The DNA extraction process of Quercus japonica provided by the present embodiment, the specimen preserved for half a year, the specimen preserved for 2 years and the specimen preserved for 5 years is as follows:

[0084] (1) Both fresh leaves and silica gel-dried leaves were cut into 3x 3cm squares, ground into powder in liquid nitrogen, and used for DNA extraction;

[0085] (2) Add 1ml of special rinse solution (4% PVP, 2% L-Ascorbic Acid, 4% Triton-X 100, 5% β-mercatoeyhanol) to the ground powder, shake, centrifuge at 10000rpm for 1min, and discard the supernatant ( If the supernatant is viscous, repeat this step until the supernatant is colorless and has a clear boundary with the precipitate);

[0086] (3) Add 1ml of 4% CTAB extract (4% CTAB (m / v), 1.4M NaCl, 100mM Tris-HCl pH 8.0, 20mM EDTA pH 8.0, 10% (w / v) N - Lauroylsarcosine Sodium salt, 3% β-mercatoeyhanol, 50mg RNase A), put in a water bath at 60°C for 1 hour, turn it up and down every 5 minutes and mix gentl...

Embodiment 3

[0143] The total plant DNA extracted by the methods of the above-mentioned Example 1 and Comparative Examples 1-3 was selected from a silica gel sample and a fresh sample, and the total DNA of the plant specimen in Example 2 was purified by the silica gel adsorption nucleic acid method, and the specific steps were as follows:

[0144] (1) Weigh 0.8g of silica gel (silica) powder, add ultrapure water to shake and mix, let it stand for 15min, centrifuge at 13200rpm for 30s and remove the supernatant (repeat the above steps 3 times); in the silica gel powder washed 3 times Add about 2.5ml of ultrapure water to make a silica gel suspension (liquid silica stock; LSK), mix the LSK, and dispense 100 μL to each tube;

[0145] (2) Take 1 tube of LSK, add 1ml of ultrapure water, shake and mix, centrifuge at 13200rpm for 20s, and discard the supernatant;

[0146] (3) Add 700 μL of 6M potassium iodide (KI) to LSK, shake and mix, add 50 μL of DNA (about 100 ng / μL of DNA), place on a small ...

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Abstract

The invention discloses an extraction and purification method for a fresh fagaceae plant sample and a fagaceae plant specimen. According to the extraction method, before nuclear lysis, a special rinsing solution containing ascorbic acid, Triton-X 100 and the like is added, so that not only can impurities such as polysaccharide and polyphenol be effectively removed to make obtained DNAs not viscousany longer, but also pigment pollution can be effectively removed to endow the extracted DNAs with high purity and high yield; compared with the step sequence of removing the impurities after the nuclear lysis, the step sequence adopted by the extraction method provided by the invention has the advantages of a better effect, and the extraction method is applicable to a later molecular experiment;according to the purification method, a self-prepared silica gel adsorption solution prepared from silica gel powder is used, and according to the purification method, the DNAs can be effectively purified, the high-purity DNAs can be obtained, and downstream PCR and enzyme digestion experiments can be met. According to the extraction and purification method, difficulties of the conventional CTABmethod and the conventional kit in fagaceae plant extraction are overcome; the extraction and purification method can be used for extracting DNAs of fresh fagaceae leaves, dry silica gel samples and dried plant specimens with relatively long preservation time and is applied to high-throughput sequencing.

Description

technical field [0001] The invention relates to a method for extracting and purifying plant DNA, in particular to a method for extracting and purifying DNA from Fagaceae plant samples rich in polysaccharides and polyphenols, especially fresh samples, silica gel dried samples and specimen samples. Background technique [0002] Most high-throughput sequencing technologies have the advantages of large data volume, wide versatility, and high resolution, and have been widely used in germplasm identification (Lai, Li et al.2010), genetic diversity analysis (Xia, Guo et al.2009 ), genetic map construction (Huang, Feng et al.2009), etc., have become important research tools and means for tree genetics and molecular breeding. [0003] Fagaceae plants are an important component of temperate and subtropical forests in the northern hemisphere, with 8 genera and about 1047 species (Govaerts and Frodin 1998). There are 7 genera and more than 300 species in our country. They are naturally...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 郑斯斯李颖李谦盛邓敏黄清俊
Owner SHANGHAI INST OF TECH
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