title: Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.
abstract: Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.
authors.affiliation: Department of Microbiology and Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824-1312, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: LaMontagne
authors.forename: F C
authors.initials: FC
authors.lastname: Michel
authors.forename: P A
authors.initials: PA
authors.lastname: Holden
authors.forename: C A
authors.initials: CA
authors.lastname: Reddy
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001673:Biodegradation, Environmental; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004783:Environmental Microbiology; D005069:Evaluation Studies as Topic; D006812:Humic Substances / Q000032:analysis; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D025341:Principal Component Analysis; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012037:Refuse Disposal
chemical_list: D004269:DNA, Bacterial; D006812:Humic Substances; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S