DNA extraction method suitable for structural analysis of microbial community in sediment

A technology for microbial community and structure analysis, applied in the field of DNA extraction for microbial community structure analysis in sediments, can solve problems such as high cost, difficult PCR amplification, and inability to effectively remove, and achieves the effect of reducing experimental costs and easy operation.

Inactive Publication Date: 2010-04-21
HOHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some of the DNA extraction methods used by most scholars cannot effectively remove the interfering substances such as humic acid in the sediment, which brings difficulties to the subsequent PCR amplification, and some require the use of high-cost nucleic acid adsorption columns.

Method used

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  • DNA extraction method suitable for structural analysis of microbial community in sediment
  • DNA extraction method suitable for structural analysis of microbial community in sediment
  • DNA extraction method suitable for structural analysis of microbial community in sediment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Weigh 0.5g sediment sample (wet weight) into a 5mL sterilized centrifuge tube, add 3mL TENPbuffer, vortex for 8min, centrifuge at 10000rpm for 5min, discard the supernatant, add 3mL TENPbuffer to the soil particle sedimentation, press Wash twice more by the above method, and finally add 3mL of PBS buffer. The pretreated sediment samples were placed at -80°C and 65°C for three times to freeze and thaw repeatedly, and lysozyme (final concentration was 1 mg / mL) was added to the samples, and placed in a water bath at 37°C for 1 h. Add proteinase K (final concentration: 0.2mg / mL) and sodium dodecylsulfonate (SDS) (final concentration: 1%, w / v) to the sample, and place in a water bath at 55°C for 1.5h (light every 10-15min) Gently invert the centrifuge tube several times to mix). Centrifuge at 10,000 rpm for 5 min at room temperature, take out 500 μL of the supernatant and place it in a new centrifuge tube, add an equal volume of phenol:chloroform:isoamyl alcohol mixture (vo...

Embodiment 2

[0031] The DNA extracted in Example 1 is used as the template of the polymerase chain reaction (PCR), using the PCR amplification instrument of the U.S. Bio-Rad company, adopting the universal primer pair GC-F341 and R518 of the 16S rRNA gene V3 region of bacteria For amplification, the specific sequence of the primers is: F341 (5'-CCTACGGGAGGCAGCAG-3'); R518 (5'-ATTACCGCGGCTGCTGG-3'), GC hairpin structure (5'-CGCCCGCCGCGCGCG GCGGGCGGGGCGGGGGCACGGGGGC-3'). (Provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd.)

[0032] The 50 μL PCR reaction system includes: DNA template 50ng, forward and reverse primers 20pmol each, 200μM dNTP, 10×PCR buffer (without MgCl 2 )5μL, 1.5mM MgCl 2 , 1U of ExTaq DNA polymerase and an appropriate amount of double distilled water to make up 50 μL.

[0033] The PCR reaction adopts the landing PCR strategy, namely: 94 °C pre-denaturation for 5 min, the first 20 cycles are 94 °C for 1 min, 65-55 °C for 1 min and 72 °C for 3 min (the...

Embodiment 3

[0035] Weigh 0.5g sediment sample (wet weight), add 5mL DNA extraction buffer (100mM Tris-HCl, pH8.0, 100mM EDTA, pH8.0, 100mM Na 3 PO 4 Buffer, pH8.0, 1.5M NaCl, 1% CTAB, w / v) and proteinase K (final concentration: 200 μg / mL), mix well, shake horizontally (37°C, 225rpm) in a shaker for about 30min, Add 1mL TE (10mM Tris-HCl, 1mM EDTA, pH8.0) and lysozyme (final concentration: 1mg / mL), continue to shake for 30min, then add 0.5mL 20% (W / V) SDS, put in a 65℃ water bath After 30 minutes, add 2% PVPP (w / v), centrifuge at 10,000 rpm for 10 minutes, transfer the supernatant to a new centrifuge tube, and repeat the extraction of the precipitate twice. The steps are: add DNA extraction buffer and 20% of SDS (w / v), water bath at 65°C for 10min, centrifuge at 10000rpm for 10min, collect the supernatant extracted three times in the same centrifuge tube, add RNaseA (final concentration is 200μg / mL), act at 37°C for 15min, add Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1)...

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Abstract

The invention discloses a DNA extraction method suitable for structural analysis of microbial community in sediment, which comprises the following steps: washing a sediment sample with TENPbuffer, and suspending the washed sediment sample in PBS buffer; repeatedly freezing and melting the pretreated sediment sample, adding muramidase, adding proteinase K and sodium dodecyl sulfate into the sediment sample, centrifuging the sample at room temperature, taking supernate, adding mixed solution of phenol, chloroform and isoamylol in equal volume into the supernate, mixing evenly by oscillation, centrifuging the obtained sample at room temperature, taking a liquid phase, adding mixed solution of chloroform and isoamylol in equal volume into the liquid phase, centrifuging the obtained sample at room temperature, taking a liquid phase, and adding precooled isopropanol into the liquid phase; centrifuging the obtained sample to remove supernate, adding precooled ethanol into the sediment, washing the sediment for three times, and centrifuging to remove the ethanol; and naturally air-drying the sample, and dissolving DNA with TE buffer solution for preservation.

Description

technical field [0001] The invention belongs to the technical field of environmental microbial ecology, and in particular relates to a DNA extraction method suitable for analyzing the microbial community structure in bottom mud. Background technique [0002] The sediment in rivers or lakes is an important part of the ecosystem, and it is also the habitat of a large number of microbial groups, which play an important role in the evolution and succession of lakes and the cycle of nutrient elements. At present, the research on the composition of microbial community in sediment has become a hot field in the research of microbial ecology. However, because many microorganisms in nature are not cultivable, it is difficult to quickly and comprehensively study the microorganisms in lake sediments by relying on traditional microscope observation and isolation culture to study microorganisms. In recent years, with the development of molecular biology techniques, some new microbial eco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 赵大勇
Owner HOHAI UNIV
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