title: Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles.
abstract: The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.
authors.affiliation: Department of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou, Guangdong, China. eesslp@mail.sysu.edu.cn
authors.forename: Lianpeng
authors.initials: L
authors.lastname: Sun
authors.forename: Xiong
authors.initials: X
authors.lastname: Ouyang
authors.forename: Yueheng
authors.initials: Y
authors.lastname: Tang
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.forename: Ying
authors.initials: Y
authors.lastname: Luo
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012722:Sewage / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage