不同提取缓冲液对红树林土壤DNA提取品质的影响

doi:10.11978/j.issn.1009-5470.2011.02.087

Journal of Tropical Oceanography ›› 2011, Vol. 30 ›› Issue (2): 87-93.doi: 10.11978/j.issn.1009-5470.2011.02.087cstr: 32234.14.j.issn.1009-5470.2011.02.087

• Mangroves • Previous Articles Next Articles

Evaluation of different extraction buffers on the quality of mangrove soil DNA

JIANG Yun-xia 1, ZHENG Tian-ling 2,3

1. Department of Environmental Health, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China; 2. Ministry of Education of Key Laboratory for Coast and Wetland Ecosystem, School of Life Sciences, Xiamen Uni-versity, Xiamen 361005, China; 3. State Key Laboratory for Marine Environmental Science, Xiamen University, Xiamen 361005, China

Received:2010-03-25 Revised:2010-05-19 Online:2011-05-06 Published:2011-05-04
Contact: 郑天凌(1955—), 男, 福建省厦门市人, 教授, 博士生导师, 主要从事环境微生物研究。E-mail: wshwzh@xmu.edu.cn E-mail:wshwzh@xmu.edu.cn
About author:蒋云霞(1973—), 女, 湖北省武汉市人, 讲师, 博士, 主要从事环境微生物研究。E-mail: jiangyxia@yahoo.com.cn
Supported by:
国家高技术研究发展计划项目(2008AA09Z408);国家自然科学基金项目(40930847、30930017、30940002); 深港创新圈项目(08Lh-04); 南方医科大学公共卫生与热带医学学院院长基金项目(GW200801-039)

Abstract

Abstract:

The authors compared the influence of six different extraction buffers on DNA yields, DNA purity, co-extracted humic compound yields, and their PCR (polymerase chain reaction) sensitivity from a typical acidic and high organic content soil type, mangrove soil. Results showed that the quality of the soil DNA obtained using the acidic SDS (sodium dodecyl sul-fate) buffer was poor. The DNA yields obtained using the PVP (polyvinylpyrrolidone) buffer were higher than the other buff-ers, whereas the co-extracted humic compound yields were significantly higher. PCR results showed that no expected bands were obtained even in 1:1000 dilutions of the DNA extracts obtained using the acidic SDS buffer or PVP buffer. On the other hand, although the PVPP (polyvinylpolypyrrolidone) buffer and the CTAB (cetyltrimethylammonium bromide) and PVPP buffer resulted in a lower DNA yield, the purity of the soil DNA was improved. In addition, the purity was improved as the concentration of PVPP in the buffer increased. Moreover, PCR amplification was successfully conducted in the 1:10 dilutions of the DNA extracts obtained using the CTAB and PVPP buffer and in the 1:100 dilutions of DNA extracts obtained using the 2% PVPP buffer, respectively. In conclusion, acidic SDS buffer and PVP buffer were not suitable for extracting DNA from the acidic and high organic content soil type; however, 2% PVPP buffer and CTAB and PVPP buffer improved the DNA quality.

Key words: microbial diversity, DNA extraction, DNA yield, humic compound, extraction buffer

JIANG Yun-xia ,ZHENG Tian-ling ,. Evaluation of different extraction buffers on the quality of mangrove soil DNA[J].Journal of Tropical Oceanography, 2011, 30(2): 87-93.

References

[1] SHEU C, WU C Y, CHEN S C, et al. Extraction of DNA from soil for analysis of bacterial diversity in transgenic and nontransgenic papaya sites[J].J Agr Food Chem, 2008, 56(24): 11969-11975.

[2] CAMPBELL J H, CLARK J S, ZAK J C. PCR-DGGE comparison of bacterial community structure in fresh and archived soils sampled along a Chihuahuan Desert elevational gradient[J]. Microbial Ecology, 2009, 57(2): 261-266.

[3] ROBINSON C H, SZARO T M, IZZO A D, et al. Spatial distribution of fungal communities in a coastal grassland soil [J]. Soil Biol Biochem, 2009, 41: 414-416.

[4] THAKURIA D, SCHMIDT O, SIURTAIN M M, et al. Importance of DNA quality in comparative soil microbial community structure analyses[J]. Soil Biol Biochem, 2008, 40(6): 1390-1403.

[5] MILLER D N, BRYANT J E, MADSEN E L, et al. Evaluation and optimization of DNA extraction and purification procedures for soil and sediments samples[J].Appl Environ Microbiol, 1999, 65(11): 4715-4724.

[6] ROBE P, NALIN R, CAPELLANO C, et al. Extraction of DNA from soil[J]. Eur J Soil Biol, 2003, 39(4):183-190.[7] SAGOVA M, CERMAK M, NOVOTNA L. Innovative methods for soil DNA purification tested in soils with widely differing characteristics[J]. Appl Environ Microbiol, 2008, 74(9): 2902-2907.

[8] THAKURIA D, SCHMIDT O, MAC SIÚRTÁIN M, et al. Importance of DNA quality in comparative soil microbial community structure analyses[J]. Soil Biol Biochem, 2008, 40(6): 1390-1403.

[9] HE J, XUA Z, HUGHES J. Pre-lysis washing improves DNA extraction from a forest soil[J]. Soil Biol Biochem, 2005, 37(12): 2337-2341.

[10] VOLOSSIOUK T, ROBB E J, NAZAR R N. Direct DNA extraction for PCR-mediated assays of soil organisms[J]. Appl Environ Microbiol, 1995, 61(11): 3972-3976.

[11] ZIPPER H, BUTA C, LAMMLE K. Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments[J]. Nucleic Acids Res, 2003, 31(7): 39.

[12] DELONG E F. Archaea in coastal marine environments[J]. Proc Natl Acad Sci, 1992, 89(12): 5685-5689.

[13] PICARD C, PONSONNET C, PAGET E, et al. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction[J]. Appl Environ Microbiol, 1992, 58(9): 2717-2722.

[14] KRSEK M, WELLINGTON E M H. Comparison of different methods for the isolation and purification of total community DNA from soil[J]. J Microbiol Methods, 1999, 39(1): 1-16.

[15] ZHOU J, BRUNS M A, TIEDJE J M.DNA recovery from soils of diverse composition [J]. Appl Environ Microbiol, 1996, 62(2): 316-322.

[16] HOLBEN W E, JANSSON J K, CHELM B K. DNA probe method for the detection of specific microorganisms in the soil bacterial community[J]. Appl Environ Microbiol, 1988, 54(3): 703-711.

[17] ORSINI M, ROMANO-SPICA V. A microwave-based method for nucleic acid isolation from environmental sam-ples[J]. Lett Appl Microbiol, 2001, 33(1): 17-20.

[18] STEFFAN R J, GOKSOYR J, BEJ A K, et al. Recovery of DNA from soils and sediments[J]. Appl Environ Microbiol, 1988, 54(12): 2908 -2915.

[19] LOOMIS W D, BATTAILE J. Plant phenolic compounds and the isolation of plant enzymes[J]. Phytochemistry, 1966, 5: 423-428.

[20] MALIYAKAL J D. An efficient method for isolation of RNA and DNA from plants containing polyphenolics[J]. Nucleic Acids Res, 1992, 20(9): 2381.

[21] TSAI Y L, OLSON B H. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction[J]. Appl Environ Microbiol, 1992, 58(2): 2292-2295.

[22] TEBBE C C, VAHJEN W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast[J]. Appl Environ Microbiol, 1993, 59(8): 2657-2665.

[23] DONG DEXIAN, YAN AN, LIU HAIMING, et al. Removal of humic substances from soil DNA using aluminium sulfate[J]. J Microbiol Methods, 2006, 66(2): 217-222.

[24] FORTIN N, BEAUMIER D, LEE K, et al. Soil washing improves the recovery of total community DNA from polluted and high organic content sediments[J]. J Microbiol Methods, 2004, 56(2): 181-191.

Evaluation of different extraction buffers on the quality of mangrove soil DNA

PDF (PC)

Like

Knowledge

Cited

Abstract

Cite this article

share this article

References

Related Articles 0

Metrics

Comments

Recommended 0