从白蚁肠道微生物(Zootermopsis Angusticollis)中提取的DNA和可视化肠道微生物

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Summary

该视频演示了居住在白蚁后肠微生物物种中提取DNA的技术。湿挂载幻灯片,这是有益的肠道微生物群落的可视化的准备工作也说明,并通过参观物种丰富的肠道环境。

Cite this Article

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Matson, E., Ottesen, E., Leadbetter, J. Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes. J. Vis. Exp. (4), e195, doi:10.3791/195 (2007).

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Abstract

白蚁是在已知有能力只消耗木材为生少数动物。白蚁肠道道包含一个高密度和物种丰富的微生物种群,在木质纤维素降解助攻主要成醋酸,助长白蚁代谢的关键营养素(Odelson Breznak,1983年)。在这些微生物种群是细菌,产甲烷古菌,在一些白蚁(“低”),真核细胞原生动物。因此,白蚁是优秀的科研课题研究微生物物种和许多他们执行他们的主人的利益的生化功能之间的相互作用。白蚁胆量,以及参与各种生化过程的关键基因的微生物种群的物种组成已探讨利用分子生物学技术(工藤等人,1998年,施密特,瓦格纳等,2003; Salmassi利百特,2003年)。这些技术依赖于从白蚁肠道内环境的高品质核酸的提取和纯化。此影片中所描述的提取工艺是从环境样品(铁道部等,1994核酸的提取和纯化的开发协议的修改编译;。Berthelet等,1996;普尔蒂等,1996;。Salmassi利德贝特2003年。Ottesen等; 2006年),它产生的DNA为模板,利用聚合酶链反应(PCR)从白蚁肠材料。

Protocol

白蚁的整个肠道DNA提取的程序总结为:

  1. 冰浴白蚁,清除肠道使用无菌镊子和稳定肠道样本缓冲液中。
  2. PVPP / SDS /酚​​缓冲区均质样品。
  3. 使用Qiagen公司DNeasy列从原油裂解的DNA提取和纯化。

协议:

  1. 在冰上,消除白蚁用无菌镊子从工人等级的胆量。
  2. 立即转移的胆量和内容,无菌,无核酸管内含有50微升冰冷1X分子生物学级TE缓冲液(1毫米的Tris - HCl,0.1 mM的EDTA,pH值8.0)。冻结在-20 ° C的样本,或进行直接与同质化。
  3. 肠道样品和缓冲传输无菌,无核酸酶2毫升螺杆上限管预装500毫克无菌氧化锆/二氧化硅珠(0.1毫米)和700μl1X TE缓冲液液,含有1%W / V polyvinylpolypyrrolidone(PVPP )。
  4. 加入50μL20%十二烷基硫酸钠(SDS),苯酚样品和500μL。
  5. 最高的设置使用三个周期同质化的30秒和30秒冰寒蝉均质(珠节拍)。
  6. 泥沙不溶性物质,在8000 X克1分钟。
  7. 净化的水层(最上层)原油裂解净化描述的方法使用Qiagen公司DNeasy列300μL等份。
  8. 量化核酸含量在-20 ° C和冻结样品,以备后用。

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Discussion

根据我们的经验,从木材喂养白蚁喜欢Zootermopis nevadensis的微生物群落中提取的DNA PCR模板充分一轮的提取和纯化后的纯。然而,一些白蚁,如乱抛垃圾的喂养和土壤哺育物种可能有较高浓度的腐殖酸在其肠道内容,可能需要额外的净化肠道微生物DNA。总DNA的产量从5 Z。nevadensis工人的胆量是在10-30微克的范围。白蚁明显小于或大于这个物种,可能需要更多或更少的标本,获得了类似的DNA量。

这种方法可以很容易地适应,让RNA提取。 RNA的提取,替代自Qiagen(catlog。76506)1X RNAprotect细菌试剂,该协议的第2步中描述的冰冷的TE缓冲。 Qiagen公司的RNeasy试剂​​和列(catlog。74104),应使用DNeasy净化上文所述的程序进行。由于DNA可从RNAprotect稳定样品纯化和只有300μL的约。 700μL的水层在第7步中检索所需的核酸纯化,这种方法可用于检索从单个样本平行的DNA和RNA。

这些技术依赖于从白蚁肠道内环境的高品质核酸的提取和纯化。此影片中所描述的提取工艺是从环境样品(更多等,1994核酸的提取和纯化的开发协议的修改编译;。Berthelet等,1996;普尔蒂等,1996;。Salmassi利德贝特2003年。Ottesen等; 2006年),它产生的DNA为模板,利用聚合酶链反应(PCR)从白蚁肠材料。

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Materials

Name Type Company Catalog Number Comments
PVPP/SDS/phenol Buffer homogeneization buffer
DNeasy Tissue Kit Kit Qiagen 77607 Used according to the protocol for isolation of genomic DNA from crude lysates (Appendix H, product manual version: July 2003)
TE Buffer Sigma-Aldrich T9285 1x buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) from 100x concentrate
zirconia/silica beads Supplies Biospec Products 11079101z 0.1 mm
PVPP Reagent Sigma-Aldrich P6755 1% w/v polyvinylpolypyrrolidone prepared from dry reagent as a 1x suspension in TE buffer
Zootermopsis nevadensis Animal Termites
SDS Reagent Sigma-Aldrich L4390 Sodium dodecyl sulfate 20% soln. in water from dry reagent
Phenol Reagent Sigma-Aldrich 77607 TE-saturated, ~73%
MiniBeadbeater-8 Tool Biospec Products 963
BSS Buffered Salt Solution, pH 7.2 Formulation per liter: 2.5 g K2HPO4, 1.0 g KH2PO4, 1.6 g KCl, 1.4 g NaCl, 0.075 g CaCl, 1 g MgCl, and 10 mL of a 1M soln. of NaHCO3
AxioPlan-2 Microscope Carl Zeiss, Inc. Outfitted with 40x objective, 1.6x optivar and 10x ocular lenses. Samples were viewed using phase contrast illumination

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References

  1. Berthelet, M., Whyte, L. G., Greer, C. W. Rapid, Direct Extraction of DNA from Soils for PCR Analysis using Polyvinylpolypyrrolidone Spin Columns. FEMS Microbiol. Lett. 138, 17-22 (1996).
  2. Kudo, T., Ohkuma, M., Moriya, S., Noda, S., Ohtoko, K. Molecular Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Hindgut of the Termite, Reticulitermes Speratus, without Cultivation. Extremophiles. 2, 155-161 (1998).
  3. Moré, M. I., Herrick, J. B., Silva, M. C., Ghiorse, W. C., Madsen, E. L. Quantitative Cell Lysis of Indigenous Microorganisms and Rapid Extraction of Microbial DNA from Sediment. Appl. Environ. Microbiol. 60, 1572-1580 (1994).
  4. Odelson, D. A., Breznak, J. A. Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites. Appl. Environ. Microbiol. 45, 1602-1613 (1983).
  5. Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. Microfluidic Digital PCR Enables Multigene Analysis of individual Environmental Bacteria. Science. 314, 1464-1467 (2006).
  6. Purdy, K. J., Embley, T. M., Takii, S., Newdell, D. B. Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method. Appl. Environ. Microbiol. 62, 3905-3907 (1996).
  7. Salmassi, T. M., Leadbetter, J. R. Analysis of Genes of Tetrahydrofolate-Dependent Metabolism from Cultivated Spirochaetes and the Gut Community of the Termite Zootermopsis Angusticollis. Microbiology. 149, 2529-2537 (2003).
  8. Schmitt-Wagner, D., Friedrich, M. W., Wagner, B., Brune, A. Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp). Appl. Environ. Microbiol. 69, 6007-6017 (2003).
  9. Tsai, Y. L., Olson, B. H. Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction. Appl. Environ. Microbiol. 58, 2292-2295 (1992).

Comments

10 Comments

  1. can you tell me the resolution power of the microscope at which you have shown the video and the make of the mcroscope used.

    Reply
    Posted by: Anonymous
    September 17, 2007 - 12:56 AM
  2. I would also like to know the magnification used to view the microbes and if any phase contrast was used with the microscope setup. Thanks.

    Reply
    Posted by: Anonymous
    December 20, 2007 - 6:24 PM
  3. As indicated in the updated protocol, a Zeiss AxioPlan-² Imaging microscope was used to aquire the images of termite gut microorganisms using Phase contrast illumination. Our particular set up allows magnification of samples up to 1,600X. This is accomplished using a 100X oil immersion objective lens + a 1.6X optivar + a 10X ocular lens. However, the images in the video use a 40X hi-dry phase contrast objective lens + the 1.6X optivar. To make the video, the videographer replaced the eyepiece with an adapter and attached the camera to that. Because we did not use a scale bar in these videos I do not know the magnification at which the camera recorded so the final magnification will depend on that factor + the window size used to view the video. I will say that the resolution is approximately consistent with what I observe at 640X magnification.

    Reply
    Posted by: Eric M.
    January 14, 2008 - 1:37 AM
  4. hello sir sir i am a project fellow in IIIM in india i want to know manual method how to isolate total DNA from termite and beetle gut(wood borer).sir kindly if you can provide me protocol for this i will be very thankful to you as i am in very need of it. thanking you sir and waiting for your reply

    Reply
    Posted by: Anonymous
    July 17, 2008 - 8:18 AM
  5. Hello,sir.I am interest in the manual DNA extraction methods from organic tissue,and acquire the best concentraction of DNA, can you provide me for this protocol, thank you.

    Reply
    Posted by: Anonymous
    September 20, 2009 - 6:21 AM
  6. The information should appear below the video on the JOVE website, and a PDF of the protocol should be downloadable, however, I notice that the link dŒs not currently work. An alternative path to the document can be found by searching the article on PubMed (www.ncbi.nlm.nih.gov/sites/entrez) and choosing the full-text article from PubMed central. I've contacted JOVE to restore the file on their website. Several strategies for purifying insect gut-community DNA exist. A crucial step in any extraction procedure is to remove PCR-inhibiting substances if present. We have found that 1% w/v polyvinylpolypyrrolidone works well on DNA derived from termite gut contents.

    Reply
    Posted by: Anonymous
    October 5, 2009 - 3:14 AM
  7. good job! very cool!

    Reply
    Posted by: jacqui s.
    December 12, 2009 - 7:28 AM
  8. Hi! I'm korean student. I want to see this full video.then what can i do?

    Reply
    Posted by: Hyun Y.
    October 31, 2014 - 9:47 AM

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