Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study.
Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.
Ning
Jing
J
Agrosphere Institute (ICG-4), Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. j.ning@fz-juelich.de
Liebich
Jost
J
Kästner
Matthias
M
Zhou
Jizhong
J
Schäffer
Andreas
A
Burauel
Peter
P
D001431:Bacteriological Techniques / Q000379:methods*; D044822:Biodiversity; D004269:DNA, Bacterial / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006812:Humic Substances / Q000032:analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic / Q000592:standards; D015203:Reproducibility of Results; D012988:Soil Microbiology
D004269:DNA, Bacterial; D006812:Humic Substances; D011933:Reagent Kits, Diagnostic