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Method for extracting nucleic acid by using magnetic nanoparticles and application thereof

A magnetic nanoparticle and extraction method technology, which is applied in the field of molecular biology, can solve the problems of complex extraction process, numerous steps, and unsatisfactory extraction effect of the kit, and achieves the effect of high extraction efficiency and no need for complex equipment.

Inactive Publication Date: 2014-06-04
陆欣华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The operation is also relatively complicated, the yield is low, and it is not easy to realize automatic operation
[0005] There are also some commercial magnetic bead extraction kits, based on the ultra-high specific surface area of ​​nano-magnetic beads, using physical properties such as electrostatic attraction to achieve nucleic acid separation and purification, but the extraction process of these kits is still relatively complicated, with many steps, and has not yet been developed. There are products specifically for miRNA extraction, and the extraction effect of existing kits is not satisfactory

Method used

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  • Method for extracting nucleic acid by using magnetic nanoparticles and application thereof
  • Method for extracting nucleic acid by using magnetic nanoparticles and application thereof
  • Method for extracting nucleic acid by using magnetic nanoparticles and application thereof

Examples

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Embodiment 1

[0031] This embodiment provides a method for extracting nucleic acid by magnetic nanoparticles, such as figure 1 As shown (serum samples do not need to add lysis buffer), it is used for the extraction of endogenous miRNA in serum, including the following steps:

[0032] Step 1: Take 100 μL of serum sample, add 100 μL of binding solution buffer (comprising components: NaCl 3M, guanidine isothiocyanate 0.05M, disodium hydrogen phosphate-citric acid buffer to adjust the pH of the binding buffer to 4), 40 μL Nano-magnetic beads with a solid content of 0.5% (polystyrene core-shell magnetic beads modified with carboxyl groups on the surface), shake and mix well, place for 10 minutes, place on a magnetic stand for magnetic separation, let stand for 15 seconds, and remove the liquid;

[0033] Step 2, add 100 μL of washing buffer (comprising components: NaCl 1M, guanidine isothiocyanate 0.05M, ethanol 20v%, phosphate buffer to adjust the pH of the washing buffer to 5), shake and mix we...

Embodiment 2

[0041] This embodiment provides a method for extracting nucleic acid by magnetic nanoparticles, and the extraction efficiency is tested using serum exogenous miRNA as a standard.

[0042] Prepare cel-mir-39 standard solution with three concentration gradients as follows:

[0043] Solution a: cel-mir-39 100pM

[0044]Solution b: cel-mir-39 10pM

[0045] Solution c: cel-mir-39 1 pM.

[0046] Take 30 μL of solutions a, b, and c and mix them into 100 μL of human serum, and follow the steps in Example 1 to obtain 30 μL of extraction products A, B, and C. 6 samples were subjected to the same RT-qPCR operation at the same time, and the operation was repeated once, and the obtained data were as follows: figure 2 (Curve a in the figure: triangle mark, b: cross mark, c: rectangle mark, A: circle mark, B: rhombus mark, C: no mark) and Table 2.

[0047] Table 2

[0048] Ct value sample cel-mir-39 a 18.56 a 19.07 A 20.22 A 19.44 b 22.17 ...

Embodiment 3

[0051] This embodiment provides a method for extracting nucleic acid by magnetic nanoparticles, such as figure 1 As shown, for the extraction of endogenous nucleic acid in cultured cells, the steps are as follows:

[0052] Step 1, collect cultured cells (<10 to the 7th power), centrifuge, remove supernatant, add lysis buffer (containing components: sodium lauryl sulfate 0.05wt%, guanidine isothiocyanate 0.5M, protease k 800μg / mL, EDTA 10mmol / L, NaCl 150mmol / L) 50μL, after the cells are lysed, add 400μL water to dilute;

[0053] Step 2, add 50 μL of binding solution (comprising components: NaCl 3M, guanidine isothiocyanate 0.05M, disodium hydrogen phosphate-citric acid buffer to adjust the pH of the binding buffer to 4), 50 μL of nanometer with a solid content of 0.5% For magnetic beads, vortex to mix and place for 10 minutes, place on a magnetic stand for magnetic separation, let stand for 15 seconds, and remove the liquid;

[0054] Step 3, add 100 μL of washing buffer (comp...

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Abstract

The invention provides a method for extracting nucleic acid by using magnetic nanoparticles and an application thereof. The method comprises the following steps: I, cracking a sample by using a cracking buffer liquid to obtain a sample cracking liquid; II, adding 5-100 microliters of liquid of magnetic nanoparticles into 20-100 microliters of the obtained sampling cracking liquid, adding 20-500 microliters of combined buffer liquid, uniformly mixing, transferring into a centrifugal tube, keeping on a magnetic frame for 10-50 seconds, and removing the liquid after nucleic acid is adsorbed and separated by the magnetic nanoparticles to obtain magnetic nanoparticles adsorbed with the nucleic acid; III, adding 50-200 microliters of washing buffer liquid into the magnetic nanoparticles adsorbed with the nucleic acid, uniformly mixing, keeping on the magnetic frame for 10-50 seconds, washing at least once, and removing the liquid; and IV, adding 20-50 microliters of elution buffer liquid into the washed magnetic nanoparticles adsorbed with the nucleic acid, uniformly mixing, keeping on the magnetic frame for 10-50 seconds, and collecting the eluent after fully eluting to obtain the nucleic acid is in the eluent. By adopting the method, the extraction of miRNA (micro Ribonucleic Acid) is realized for the first time, and high extraction efficiency is achieved.

Description

technical field [0001] The invention relates to a nucleic acid extraction method and application of magnetic nanoparticles, belonging to the technical field of molecular biology. Background technique [0002] Contemporary biology, medical research, etc. have gone deep into the molecular level, such as the nucleic acid level, and efficient nucleic acid extraction and separation technology is particularly important. For example, the screening of miRNA tumor markers in body fluids such as serum / plasma requires a simple and efficient miRNA extraction technology. [0003] At present, the most conventional technology is the classic phenol / chloroform extraction method, which uses the principle of extraction to isolate nucleic acids. However, organic solvents such as phenol / chloroform are extremely toxic to the human body and have a negative impact on the environment. Moreover, the operation process is complicated, and the results are greatly affected by human factors, so it is dif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 陆欣华代广颖
Owner 陆欣华
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