DNA extraction method

An extraction method and sample technology, applied in the field of DNA extraction, can solve problems such as false negatives, and achieve the effect of simple extraction operation, excellent simplicity and speed, and improved sensitivity

Pending Publication Date: 2018-01-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the actual sample detection, although PCR is a mature method, how to quickly and easily obtain the DNA of the tested sample is a difficult problem
Although PCR has strong amplification ability and high sensitivity, if there are PCR inhibitors in the extracted DNA template, it will cause false negative

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Adding BSA solution into the PCR system can eliminate the inhibition of PCR by aminated magnetic nanoparticles.

[0038] Preparation of Aminated Magnetic Nanoparticles:

[0039] 1) Fe 3 o 4 Preparation of magnetic nanoparticles: 0.02mol FeCl 3 ·6H 2 O and 0.01mol FeSO 4 ·7H 2 O was dissolved in 150ml of deionized water, heated to 85°C under the protection of nitrogen, and continued to react for 25min, magnetically separated, washed and then vacuum-dried. 2) Preparation of magnetic nanoparticles: 9ml n-hexanol, 37.5mL cyclohexane, 8.85mL Triton 100, 200mg Fe 3 o 4 The nanoparticles were mixed, 0.5 mL tetraethyl orthosilicate was added, and then 0.3 mL ammonia water was added, and the reaction was performed under magnetic stirring at room temperature for 24 hours. Magnetic separation, washing and vacuum drying. 3) Amination modification: 200mg of the above-mentioned Fe 3 o 4 Nanoparticles and 1ml of 3-aminopropyltriethoxysilane were mixed in 30mL of ...

Embodiment 2

[0041] Example 2: Extract the genomic DNA of the fungal culture, amplify the gene sequence by PCR, and determine whether it is matsutake by sequencing.

[0042]Take a small amount of fungal culture (Shanghai Academy of Agricultural Sciences) and grind it three times with liquid nitrogen, weigh 200 mg sample, add 1000 μL of TE buffer solution (pH=9), add 2 μL of 500 U / mL lysozyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd. Technology Co., Ltd.), incubate at 37°C for 30min. Add 10 μL of Triton X100 (purchased from Shanghai Sinopharm Group) and 50 μL of proteinase K (purchased from Merck) at a concentration of 20 μL / mL, and incubate at 65° C. for 30 min. Centrifuge at 12000rpm for 5min, absorb the supernatant and transfer to a new 1.5mL centrifuge tube, add 40μg of the aminated magnetic nanoparticles prepared in Example 1, and react at 90°C for 10min. After TE was eluted once, the solid component was magnetically separated at the bottom of the small PCR tube, and ad...

Embodiment 3

[0044] Example 3: Genomic DNA is extracted from cottonseed hulls, and PCR is used to identify whether it is transgenic cottonseed hulls.

[0045] Take a small amount of cottonseed hulls (from Shanghai Academy of Agricultural Sciences) and grind them three times with liquid nitrogen, weigh 200 mg of the sample, add 1000 μL of TE buffer solution (pH=9), add 10 μL of Triton X100 and 50 μL of protease at a concentration of 20 μL / mL K, 65°C for 30 minutes. Then, treat at 100°C for 10 minutes. Centrifuge at 12000rpm for 5min, draw the supernatant into a new 1.5mL centrifuge tube, add 200μg purchased aminated magnetic beads ( Plus Amine, purchased from Bangslab Company), reacted at 90°C for 10min. After washing once with TE buffer, the solid components were magnetically separated into different PCR small tubes, and added to the PCR system (the system contained 2 μL of 10% BSA solution).

[0046] Amplification primer pairs are: CaMV35S-r / f, NOS-r / f, CpTI-r / f, Cry IA(b)+Cry IA(c)-r...

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PUM

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Abstract

The invention provides a DNA extraction method. The method comprises the following steps: suspending a sample in a TE buffer solution; performing cell lysis and DNA release; and aminating magnetic nano-particles to adsorb the DNA. According to the DNA extraction method disclosed by the invention, the detection sample is simply treated, the DNA in the sample is rapidly separted by virtue of the aminated magnetic nano-particles, and a magnetic complex is directly added into a PCR (Polymerase Chain Reaction) so as to realize rapid and sensitive detection. Moreover, the DNA extraction method is applicable to multiple detection samples.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA extraction method suitable for PCR detection. Background technique [0002] For the detection of living substances, PCR can amplify a specific DNA sequence from a few copies to millions of times in vitro in a relatively short period of time. However, in the actual sample detection, although PCR is a mature method, how to quickly and easily obtain the DNA of the sample to be tested is a difficult problem. Although PCR has extremely strong amplification ability and high sensitivity, if there are PCR inhibitors in the extracted DNA template, it will cause false negatives. In order to obtain purified DNA (DNA templates that remove PCR interfering substances as much as possible), various cumbersome purification steps are often required. [0003] Therefore, a simple and quick DNA extraction method is needed to meet the requirements of PCR detection. Contents of the invention [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
Inventor 白亚龙周昌艳索玉娟沈源源瞿阳林婷
Owner SHANGHAI ACAD OF AGRI SCI
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