Evaluation of DNA Extraction and Purification Methods for Corn Straw Biogas Slurry

Abstract:

Corn straw biogas slurry always contains humic substances, which poses particular challenges in obtaining PCR-amplifiable DNA for analysis of microbial community. To establish an efficient and reliable DNA extraction method for straw biogas slurry, four approaches: i.e., direct SDS-based method, direct bead-based method, indirect SDS-based method, and indirect bead-based method were evaluated by comparing DNA yield, humic acid contamination, PCR amplifiability, and restriction fragment length polymorphisms (RFLP) of amplified 16S rRNA genes. Direct DNA extraction methods yielded 3-fold higher amounts of DNA than indirect procedures, but its DNA purity was lower. The A₂₆₀/A₂₃₀ ratio of DNA from indirect methods (0.8-0.85) were higher than that of DNA from direct methods (0.5-0.6), indicating DNA from direct methods contained high levels of humate contamination. PCR amplification was successful with crude DNA from indirect methods, but not with crude DNA from direct methods. PCR products could also be obtained with purified DNA from direct bead-based method, whereas not direct SDS-based method. Among the four methods, direct bead-based method, indirect SDS-based method and indirect bead-based method could obtain high-quality DNA extracts from corn straw biogas slurry. RFLP analysis further demonstrated the restriction patterns of amplified 16S rRNA genes from three methods were relatively identical microbial diversity.

• Periodical: Advanced Materials Research (Volume 773)
• Pages: 113-117
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.773.113
• Online since: September 2013
• Authors: Jiang Tao Qiao, Rong Bo Guo, Xiao Shuang Shi, Yan Ling Qiu
• Keywords: Corn Straw Biogas Slurry, DNA Extraction, Molecular Ecology, PCR, RFLP

References

[1] J.J. Liu, W.X. Wu, C.J. Chen, F.Q. Sun and Y.X. Chen. Prokaryotic diversity, composition structure, and phylogenetic analysis of microbial communities in leachate sediment ecosystems[J]. Applied Microbiology and Biotechnology, 2011, 91(6): 1659-1675.

DOI: 10.1007/s00253-011-3354-8

[2] S. Weber, S. Stubner, R. Conrad Bacterial populations colonizing and degrading rice straw in anoxic paddy soil[J]. Applied and Environmental Microbiology, 2001, 67(3): 1318-1327.

DOI: 10.1128/aem.67.3.1318-1327.2001

[3] M. Klocke, P. Mähnert, K. Mundt, K. Souidi, B. Linke. Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate[J]. Systematic and Applied Microbiology, 2007, 30(2): 139-151.

DOI: 10.1016/j.syapm.2006.03.007

[4] A. Henne, R.A. Schmitz, M. Bömeke, G. Gottschalk, R. Daniel. Screening of Environmental DNA Libraries for the Presence of Genes Conferring Lipolytic Activity on Escherichia coli[J]. Applied and Environmental Microbiology, 2000, 66(7): 3113-3116.

DOI: 10.1128/aem.66.7.3113-3116.2000

[5] J.E.M. Stach, S. Bathe, J.P. Clapp, R.G. Burns. PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods[J]. FEMS Microbiology Ecology, 2001, 36(2-3): 139-151.

DOI: 10.1111/j.1574-6941.2001.tb00834.x

[6] E.M. Gabor, E.J. Vries, D.B. Janssen. Efficient recovery of environmental DNA for expression cloning by indirect extraction methods[J]. FEMS Microbiology Ecology, 2006, 44(2): 153-163.

DOI: 10.1016/s0168-6496(02)00462-2

[7] W.G. Weisburg, S.M. Barns, D.A. Pelletier, D.J. Lane. 16S ribosomal DNA amplification for phylogenetic study[J]. Journal of Bacteriology, 1991, 173(2): 697-703.

DOI: 10.1128/jb.173.2.697-703.1991

[8] J.Z. Zhou, M.A. Bruns, J.M. Tiedje. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology[J], 1996, 62(2): 316-322.

DOI: 10.1128/aem.62.2.316-322.1996

[9] M.G. LaMontagne, F.C. Michel Jr, P.A. Holden, C.A. Reddy. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis[J]. Journal of Microbiological Methods, 2002, 49(3): 255-264.

DOI: 10.1016/s0167-7012(01)00377-3