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Method for extracting free nucleic acid from large-volume cell-free body fluid

A cell-free body fluid and free nucleic acid technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of skin corrosion, human body damage, and low extraction efficiency, and achieve the effect of increasing versatility, improving efficiency, and high purity

Inactive Publication Date: 2018-03-13
SUZHOU GENEPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The phenol-chloroform extraction method is low in cost, but the extraction efficiency is low and the reproducibility is poor. Suitable for the extraction of small fragments of circulating nucleic acids

Method used

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  • Method for extracting free nucleic acid from large-volume cell-free body fluid
  • Method for extracting free nucleic acid from large-volume cell-free body fluid
  • Method for extracting free nucleic acid from large-volume cell-free body fluid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Comparison of non-concentrated and concentrated plasma cfDNA extraction

[0050] 1) Extract plasma cfDNA without concentration

[0051] 1. Take 1ml of plasma and add it to a 2ml centrifuge tube, then add 1ml of lysate and 50ul of proteinase K, mix well, resuspend the mixture and incubate at 58°C for 1 hour;

[0052] 2. Add 400ul of isopropanol to the resuspension, mix it upside down; draw 700ul of the sample into a spin column with a 2ml collection tube, lightly cover the lid, centrifuge at 12000 r / min in a high-speed centrifuge for 1min, and discard the flow-through. Pipette the remaining samples into the spin column and repeat this operation until all the samples are transferred;

[0053] 3. Add 700ul protein-removing solution to the spin column, centrifuge at 12000 r / min for 1min in a high-speed centrifuge, discard the flow-through solution, and repeat this step once;

[0054] 4. Add 700ul of 80% ethanol to the spin column, centrifuge at 12000 r / min for ...

Embodiment 2

[0063] Example 2: Optimization of the amount of precipitating agent in the process of concentrating and extracting plasma cfDNA

[0064] 1. Divide 2ml of plasma into two 2ml centrifuge tubes, add 1ml of plasma in each tube, add 70ul of precipitant and 114ul of buffer, 140ul of precipitant and 228ul of buffer, and then centrifuge each The tube was vortexed, placed at room temperature for 1 min, centrifuged at 10,000 r / min for 3 min in a high-speed centrifuge, and the supernatant was discarded;

[0065] Perform the following operations on the above two tubes of plasma respectively,

[0066] 2. Add 700ul of sterilized water to the sediment, mix it upside down three times, centrifuge with a high-speed centrifuge at 12000r / min for 3min, and discard the supernatant;

[0067] 3. Add 400ul lysate and 50ul proteinase K to the precipitate, resuspend the precipitate, and incubate at 58°C for 15min;

[0068] 4. Add 400ul of isopropanol to the resuspension, mix by inverting, draw 700ul o...

Embodiment 3

[0074] Example 3: Optimization of the amount of lysate used in the process of concentrating and extracting plasma cfDNA

[0075] 1. Divide 4ml of plasma into four 2ml centrifuge tubes, add 140ul of precipitant and 228ul of buffer solution to each tube of 1ml of plasma, vortex and mix well, place at room temperature for 1min, and centrifuge at 10000r / min for 3min in a high-speed centrifuge , discard the supernatant;

[0076] 2. Add 700ul of sterilized water to the sediment of the above four centrifuge tubes, mix by inverting three times, centrifuge at 12000r / min for 3min in a high-speed centrifuge, and discard the supernatant;

[0077] 3. Add 4 different volumes of lysate to the sediment of the above 4 centrifuge tubes: 200ul lysate, 300ul lysate, 400ul and 500ul lysate, then add 50ul proteinase K to resuspend the precipitate, Incubate at 58°C for 15 minutes;

[0078] Perform the following operations on the resuspension in the above four centrifuge tubes respectively,

[007...

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Abstract

The invention relates to a method for extracting free nucleic acid from large-volume cell-free body fluid. The method comprises the following steps: S1, concentrating, specifically, adding a precipitating agent and a buffer solution into the cell-free body fluid, evenly mixing by means of vortex and then placing at the room temperature, centrifuging the cell-free body fluid, and discarding a supernatant; S2, cracking, specifically, adding sterile water into precipitate obtained in the S1, evenly mixing upside down, centrifuging the obtained mixture, and discarding the supernatant; then, addinglysate and protein K, resuspending precipitate, and carrying out incubation at high temperature; S3, purifying, specifically, adding isopropanol into the suspension obtained in the S2, evenly mixingand then enabling the obtained mixture to be combined with an adsorption column or magnetic beads, cleaning the sample with washing liquor for a plurality of times, and finally eluting the product with eluant so as to obtain the high-purity free nucleic acid. The desoxyribonucleic acid (DNA) sample extracted by using the method is high in yield and purity and is applicable to the detection, such as fluorescence quantitative polymerase chain reaction (PCR), with higher requirement for a template, so that the universality of subsequent sample detection is improved.

Description

technical field [0001] The invention relates to the technical field of extracting nucleic acid DNA, in particular to a method for extracting free nucleic acid from a large volume of cell-free body fluid. Background technique [0002] Circulating nucleic acid is a kind of nucleic acid in the free state outside the cell. There are circulating DNA, RNA and microRNA in human plasma / serum. These nucleic acid molecules are released into the peripheral blood after apoptosis or death, and have small fragments and low content. specialty. In many disease states, such as stroke, myocardial infarction, diabetes and many malignant tumor patients, plasma free gDNA will be significantly increased. Circulating tumor DNA (ctDNA) in cancer patients comes directly from tumor cells, and its genetic analysis can obtain all the information on the malignancy of the tumor, which can provide an important basis for the early diagnosis and identification of tumors, prognosis judgment and curative eff...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 饶品彬李娜张佩琢
Owner SUZHOU GENEPHARMA
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