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DNA extracting agent, DNA extracting method and DNA extracting kit

An extraction method and kit technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of low DNA yield, affecting PCR, high-throughput sequencing, low DNA purity, etc.

Active Publication Date: 2019-05-24
RESEARCH INSTITUTE OF TSINGHUA UNIVERSITY IN SHENZHEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both the current commercial extraction kits and the traditional phenol-chloroform extraction method have their defects. For example, most of the current commercial extraction kits are column extraction methods. The shear force generated during centrifugal filtration will seriously degrade DNA and cause low DNA yield. , it is difficult to meet the requirements of metagenomic sequencing, and the cost is high; the DNA purity obtained by the traditional phenol-chloroform extraction method is low, which affects subsequent PCR, high-throughput sequencing, etc.

Method used

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  • DNA extracting agent, DNA extracting method and DNA extracting kit
  • DNA extracting agent, DNA extracting method and DNA extracting kit
  • DNA extracting agent, DNA extracting method and DNA extracting kit

Examples

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specific Embodiment

[0041] The following will be described in detail in conjunction with specific embodiments. The following examples, unless otherwise specified, do not include other components except unavoidable impurities. The drugs and instruments used in the examples are all routine choices in the art unless otherwise specified. The experimental methods for which specific conditions are not indicated in the examples are implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the manufacturer.

Embodiment 1

[0044] Weigh three stool samples from the same person, each 200 mg, and perform the following operations for each stool sample:

[0045] (1) Pretreatment: Mix the feces sample with 5 times the volume of normal saline, vortex to mix, centrifuge at 2000rpm / min at 4°C for 2min, take the supernatant and discard the precipitate, then 10000rpm / min at 4°C Centrifuge at a high speed for 1 min, discard the supernatant, and keep the centrifuged pellet. Then add 1mL PBS (pH 7.4) to the precipitate, vortex and mix well, centrifuge at 2000rpm / min for 2min at 4°C, take the centrifuged supernatant and discard the precipitate; then put the centrifuged supernatant at 4 Centrifuge at 10,000 rpm / min for 1 min at ℃, discard the supernatant, and keep the precipitate. At this time, the precipitate is the purified microbial cell, which is the sample to be lysed.

[0046] (2) Lysis solution to lyse the sample: add 800 μL of lysis solution to the sample to be lysed obtained in step (1), vortex to mix...

Embodiment 2

[0052] The steps and methods for extracting DNA in the feces in Example 2 are roughly the same as in Example 1, except that the lysate used in Example 2 is composed of guanidine isothiocyanate with a final concentration of 1M and Tris- It is composed of HCl, N-lauroyl sarcosine with a final mass volume percentage of 5%, and a phosphate buffer solution with a final concentration of 0.05M pH8.0. TENP buffer was composed of 100 mM Tris pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl and 6% PVPP in final mass volume. The first wash consisted of phosphate at a final concentration of 0.05M pH 8.0 and potassium acetate at a final concentration of 1.5M.

[0053] The concentration of DNA in three groups of stool samples of embodiment 2 is shown in table 1 respectively, and electrophoresis figure is as shown in Table 1. figure 1 shown. In Table 1, numbers 5-7 are the extraction results of the three stool samples in Example 2. figure 1 The number 1 in the middle corresponds to the E.coli DNA s...

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Abstract

The invention relates to a DNA extracting agent, a DNA extracting method and a DNA extracting kit. The DNA extracting agent includes a lysis solution and a cleaning agent; the lysis solution includesguanidinium isothiocyanate having a final concentration of 1-5 M, Tris-HCl having a final concentration of 0.01-0.2 M, and N-lauroylsarcosine having final mass percent by volume of 1-10%; the cleaningagent includes at least one of crosslinked polyvinyl pyrrolidone and polyvinyl pyrrolidone. The lysis solution and the cleaning agent in the DNA extracting agent can cooperate with each other, degradation of DNA in the extraction process can be decreased, and DNA purity can be improved; DNA prepared with the DNA extracting agent meets the requirements of metagenomics sequencing.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA extraction reagent, a DNA extraction method and a DNA extraction kit. Background technique [0002] Human health is closely related to the quantity and types of microorganisms on the surface and organs of the human body, especially intestinal microorganisms, which play an important role in human health. A large number of studies in recent years have found that a variety of microorganisms in the human gut play an important role in diseases such as immunity, development, inflammation, metabolic diseases, mental disorders, and cancer. The study of gut microbiota can help us prevent, diagnose and treat diseases. Ultimately cure or suppress such diseases. [0003] As one of the sources of intestinal microbiome research, fecal samples are convenient and non-invasive. However, human feces are complex and contain fibers, microorganisms, undigested particles, nucleases, and human exfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 何筠张琦文田晖
Owner RESEARCH INSTITUTE OF TSINGHUA UNIVERSITY IN SHENZHEN
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