title: An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis.
abstract: An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.
authors.affiliation: Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tian
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Zhang
authors.forename: Dongyang
authors.initials: D
authors.lastname: Liu
authors.forename: Tiantian
authors.initials: T
authors.lastname: Zhou
authors.forename: Qirong
authors.initials: Q
authors.lastname: Shen
authors.forename: Biao
authors.initials: B
authors.lastname: Shen
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D014644:Genetic Variation; D008372:Manure / Q000032:analysis* / Q000382:microbiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D012987:Soil / Q000737:chemistry*
chemical_list: D004269:DNA, Bacterial; D008372:Manure; D012336:RNA, Ribosomal, 16S; D012987:Soil