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Nano magnetic bead based biological sample nucleic acid extracting and purifying method

A technology of nano-magnetic beads and biological samples, which is applied in the direction of DNA preparation and recombinant DNA technology. It can solve the problems of easy loss of small fragments, long time-consuming nucleic acid, and difficulty in automation, so as to shorten the extraction and purification time and improve the nucleic acid extraction process. , easily automated effects

Inactive Publication Date: 2019-02-05
TIANJIN JIAMEI HUIZHONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 1. When organic solvents such as phenol and chloroform are used to extract and purify nucleic acids from biological samples, phenol and chloroform are often toxic and produce peculiar smells, which affect the health of operators. This method also requires multiple centrifuges, so the extraction and purification of nucleic acids The process is time-consuming and difficult to automate
[0004] 2. Although the method of extracting and purifying nucleic acid from biological samples by the column membrane method can simplify the operation process, it is easy to lose small fragments in the process of nucleic acid extraction and purification, and it still needs multiple centrifugation or negative pressure for extraction and purification, and it is also easy to cause cross-contamination
[0005] 3. As a new nucleic acid extraction method, the magnetic bead method is more and more widely used because of its simple operation, no need for negative pressure or repeated centrifugation, and easy automation. However, this method is mostly based on the existing nucleic acid extraction method. Purification steps are carried out, and the demand for initial biological samples is still hundreds of milligrams, so it is difficult to guarantee efficient extraction and purification of nucleic acids from precious or rare human tissues, biological specimens, archaeological and forensic identification samples

Method used

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  • Nano magnetic bead based biological sample nucleic acid extracting and purifying method
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  • Nano magnetic bead based biological sample nucleic acid extracting and purifying method

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1, extracting nucleic acid from a small amount of plant cells, it is mainly implemented according to the following steps:

[0067] Step 1, plant sample preparation: wash the floating soil on the surface with sterilized water, drain quickly, weigh 5-10mg young leaves, 5-10mg petals, 5-10mg seeds, 20-40mg stems or 50-100mg young roots , put it into a 1.5ml clean centrifuge tube for later use, and then carry out the following process:

[0068] (1) Add about 500 μl of plant cell lysate into 1.5ml centrifuge tube 1, assist in breaking plant cells by physical, chemical or enzymatic methods, achieve sufficient crushing and grinding, and release nucleic acid from plant sample cells, thereby obtaining lysed cells solution;

[0069] (2) Heat the 1.5ml centrifuge tube 1 in step 1 (1) in a water bath at 70°C for 10 minutes, and perform intermittent mixing;

[0070] (3) Add 100 μl of polysaccharide-protein removal solution and 100 μl of absolute ethanol to the 1.5ml cen...

Embodiment 2

[0091] Embodiment 2, extracting nucleic acid from a small amount of animal cells, it is mainly implemented according to the following steps:

[0092] Step 1, animal sample preparation: Weigh 5mg of liver, 5-10mg of muscle, or 5-10mg of heart, put them into a 1.5ml clean centrifuge tube for later use, and then carry out the following process:

[0093] (1) Add about 600 μl of animal cell lysate into 1.5ml centrifuge tube 1, assist in breaking animal cells by physical, chemical or enzymatic methods, achieve sufficient crushing and grinding, and release nucleic acid from animal sample cells, thereby obtaining lysed cells solution;

[0094] (2) Heat the 1.5ml centrifuge tube 1 in step 1 (1) in a water bath at 50°C for 10 minutes, and perform intermittent mixing;

[0095] (3) Add 120 μl of polysaccharide-protein removal solution and 120 μl of absolute ethanol to the 1.5ml centrifuge tube 1 in step 1 (2), and mix well;

[0096] (4) Place the 1.5ml centrifuge tube in step 1(3) at ro...

Embodiment 3

[0116] Embodiment three, by extracting nucleic acid from trace bacterial cells, it is mainly implemented according to the following steps:

[0117] Step 1, Bacterial sample preparation: Cultivate the bacteria overnight, centrifuge the bacterial solution at a speed of 7000-8000r / min for 3-5 minutes, discard the supernatant, take 5-10mg of bacterial cells into a clean centrifuge tube, and then proceed to the next step Described process:

[0118] (1) Add about 600 μl of bacterial cell lysate to the 1.5ml centrifuge tube 1, and assist in breaking the bacterial cells by physical, chemical or enzymatic methods to achieve sufficient crushing and grinding, so that the bacterial sample cells release nucleic acids, thereby obtaining lysed cells solution;

[0119] (2) Heat the 1.5ml centrifuge tube 1 in step 1 (1) in a water bath at 70°C for 10 minutes, and perform intermittent mixing;

[0120] (3) Add 100 μl of polysaccharide-protein removal solution and 100 μl of absolute ethanol to ...

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Abstract

The invention provides a nano magnetic bead based biological sample nucleic acid extracting and purifying method. The nano magnetic bead based biological sample nucleic acid extracting and purifying method comprises the following steps: step I, preparing and processing a biological sample, taking and putting 5-10 mg of the treated biological sample into a 1.5 ml centrifugal tube, adding cell lysisbuffer to break up cells, and separating and collecting liquid supernatant by centrifuging; step II, performing nucleic acid adsorption by adding nano magnetic bead suspension liquid into the liquidsupernatant obtained in step I, and collecting adsorbed nucleic acid-nano magnetic bead composite; and step III, purifying and eluting the nucleic acid-nano magnetic bead composite obtained in step IIto obtain a nucleic acid solution with purity of 1.7-2.1 and the yield of 9-40 mu g. The method can be adopted to extract and purify DNA or RNA biomacromolecules with good integrity, high purity andhigh yield from the biological sample within a range from several milligrams to more than a dozen milligrams based on a nano magenta bead process, so that damages on samples such as precious or rare human tissues, biological samples, archaeological samples and medicolegal expertise samples as a result of high quantity demanded on the biological sample are effectively avoided.

Description

technical field [0001] The invention relates to the technical field of extracting and purifying nucleic acid by a magnetic bead method, in particular to a method for extracting and purifying nucleic acid from a biological sample based on nano magnetic beads. Background technique [0002] Nucleic acid includes deoxyribonucleic acid (RNA) and ribonucleic acid (DNA), both of which exist in a state bound to proteins in cells. The chromosomal DNA of eukaryotic organisms is a double-stranded linear molecule, and the "chromosome", plasmid, and eukaryotic organelle DNA of prokaryotic organisms are double-stranded circular molecules; some bacteriophage DNA is sometimes single-stranded circular molecules, and RNA molecules are in most All organisms are single-stranded linear molecules, and different types of RNA molecules can have different structural characteristics. As for the DNA and RNA molecules of viruses, they exist in various forms, including double-stranded circular, single-s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 李广善
Owner TIANJIN JIAMEI HUIZHONG TECH
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