Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.
The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.
Weiss
Agnes
A
Process Biotechnology, University of Bayreuth, 95440 Bayreuth, Germany.
Jérôme
Valérie
V
Freitag
Ruth
R
D019149:Bioreactors / Q000382:microbiology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006812:Humic Substances / Q000032:analysis; D007220:Industrial Waste / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012722:Sewage / Q000382:microbiology
D006812:Humic Substances; D007220:Industrial Waste; D012722:Sewage; D004247:DNA