Method for efficiently and quickly extracting crop genome DNA (DeoxyriboNucleic Acid)

A crop and rapid technology, applied in the field of DNA extraction, can solve the problems of hindering PCR reaction, increasing the number of steps and reagents, and affecting the potential risks of PCR reaction, so as to shorten the extraction time, simplify the extraction steps, accelerate cell membrane rupture and protein denaturation.

Inactive Publication Date: 2017-10-27
HUNAN RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of the existing alkaline lysis methods only add alkaline solution for denaturation lysis, and the extracted DNA solution will hinder the subsequent PCR reaction due to the high pH; some will add HCl solution for neutralization after adding alkaline solution, but only add HCl solution There is also the potential risk of making the pH of the extracted DNA solution low, which will affect the subsequent PCR reaction; some add HCl solution and then add Tris buffer solution to adjust the pH value, and the steps and reagents are significantly increased; and the above methods need to be performed in a high-temperature water bath Complete the process of alkaline lysing cells and extracting DNA

Method used

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  • Method for efficiently and quickly extracting crop genome DNA (DeoxyriboNucleic Acid)
  • Method for efficiently and quickly extracting crop genome DNA (DeoxyriboNucleic Acid)
  • Method for efficiently and quickly extracting crop genome DNA (DeoxyriboNucleic Acid)

Examples

Experimental program
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Effect test

Embodiment 1

[0045]The extraction process of the rice variety DNA provided in this implementation is as follows: take a single rice seed and soak it for 12-24 hours, place it on a germination bed, germinate at 20°C-30°C for 4-7 days, grow a seedling of 0.5-1cm, take the seedling and add it to 96 wells In the plate, there is one seedling sample and two steel balls per hole. Add 50 μL of the mixed solution of NaOH solution and TritonX-100 solution to each well, wherein the concentration of NaOH solution is 0.1mol / L, the volume concentration of TritonX-100 solution is 1%, grind at 70Hz for 90s to obtain a homogenate; add 0.1mol / L Tris solution (pH value 8.0) was 50 μL, the supernatant was collected, and 1 μL was used for PCR reaction.

[0046] Select the SSR primers applicable to the purity identification of this variety to detect 24 seeds of this variety, and the results are as follows: figure 1 shown. figure 1 It shows that the quality of the DNA extracted by the method of the present inv...

Embodiment 2

[0048] The extraction process of the corn variety DNA provided in this implementation is as follows: take a single corn seed and place it on a germination bed, germinate at 20°C to 30°C for 4 to 7 days, grow a root system of 0.5 to 1 cm, take the root system and add it to a 96-well plate, 1 Plant root samples and 2 steel balls. Add NaOH solution and NP to each well 40 The mixed solution of the solution is 50 μL, the concentration of NaOH solution is 0.1mol / L, NP 40 The volume concentration of the solution was 1%, and it was ground at 70 Hz for 90 s to obtain a homogenate; 50 μL of 0.1 mol / L Tris solution (pH value 8.0) was added to collect the supernatant, and 1 μL was used for PCR reaction.

[0049] The SSR primers suitable for the purity identification of this corn variety were selected to detect 24 seeds of this variety, and the results were as follows: figure 2 shown. figure 2 It was shown that the quality of the extracted DNA fully met the requirements of the PCR rea...

Embodiment 3

[0051] The DNA extraction process of the rapeseed variety provided in this implementation is as follows: Put the rapeseed seeds on the germination bed, germinate at 15°C-25°C for 5-7 days, take 50-100 seedlings of the same variety with a length of 0.5-1cm, mix them, and add them together with 1 steel ball 2mL centrifuge tube. Add 500 μL of the mixed solution of NaOH solution and TritonX-100 solution to each tube, wherein the concentration of NaOH solution is 0.1mol / L, the volume concentration of TritonX-100 solution is 1%, grind at 60Hz for 60s to obtain a homogenate; add 0.1mol / L Tris solution (pH value 8.0) 500 μL, collect the supernatant, take 1 μL of extracted DNA for PCR reaction.

[0052] Using SSR primers for Brassica napus variety identification to test the authenticity of two rapeseed varieties, the results are as follows image 3 shown. image 3 It shows that the performance of the 12 pairs of SSR primer sites in the figure is not completely consistent between the ...

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Abstract

The invention discloses a method for efficiently and quickly extracting crop genome DNA (DeoxyriboNucleic Acid). The method specifically comprises the following steps: putting a root system, a seedling or a leaf which grows after the single seed of a crop sprouts into a container, and adding an alkaline solution and steel balls to carry out grinding pyrolysis so as to obtain homogenate; adding Tris buffered solution into obtained lysate, and collecting upper-layer clear solution. Compared with a traditional alkaline pyrolysis method, the method disclosed by the invention is characterized in that TritonX-100 solution or NP40 solution are added in an alkaline grinding pyrolysis process to more simply and quickly extract DNA, extraction quality is higher, and an extraction effect is better. Since the Tris buffered solution is added, an influence for subsequent PCR (Polymerase Chain Reaction) since the pH (Potential of Hydrogen) value of the extracted DNA solution is overhigh or overlow can be avoided, and only two solutions need to be added to extract high-quality DNA; a high-temperature water bath process is omitted, and the efficient extraction of the DNA can be realized only through two steps. The method established by the invention is suitable for the quick detection of the purity, the authenticity and the transgenic ingredients of the single seed of the crop and the auxiliary selection research of the key character molecular marker of the crop.

Description

technical field [0001] The invention belongs to the technical field of DNA extraction, and in particular relates to a method for efficiently and rapidly extracting genome DNA of crops, in particular, a method suitable for efficiently and rapidly extracting DNA from a single seed of crops. Background technique [0002] With the rapid development of biotechnology, molecular biology techniques based on PCR technology, such as QTLs mapping, gene cloning, molecular marker-assisted selection, and detection of transgenic plants, have been widely used in the research and breeding of key crop traits. The application is inseparable from the extraction of genomic DNA. [0003] The separation of DNA in plant tissue needs to break the barrier of the cell wall, release the DNA from the cell membrane, and separate it from biological macromolecules such as proteins, polysaccharides, and fats. However, currently commonly used DNA extraction methods, such as CTAB method, SDS method, phenol m...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/119C12Q2527/125
Inventor 刘之熙朱克永
Owner HUNAN RICE RES INST
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