Improving the recovery of qPCR-grade DNA from sludge and sediment.
DNA extraction is often considered as the limiting step of most molecular approaches in ecology and environmental microbiology. Ten existing DNA extraction protocols were compared for recovery of DNA from sludge and a modified version of the protocol described by Porteous et al. (Mol Ecol 6:787-791, 1997) was determined to be the best method for recovery of DNA suitable for PCR. In this respect, it appeared that the commonly used guanidine isothiocyanate could impair the quality of the extracted DNA unless its concentration is lowered. Second, conditioning the samples as liquors as opposed to pellets critically impacts the outcome of the extraction. The suitability of the modified Porteous protocol for quantitative PCR applications is demonstrated in a series of experiments showing the absence of interfering coextracted inhibitors and the linear correspondence between the concentrations of input target DNA and PCR product. Interestingly, it is also shown that the nature of the environmental matrices affects the recovery yield of both circular plasmids and chromosomal DNA, resulting in an apparent fluctuation of the plasmid copy number per cell. This means that quantitative data obtained by PCR remain comparable as long as they apply to an identical target sequence extracted from a similar environment and amplified under the same conditions.
Bonot
Sébastien
S
UMR CNRS-Nancy Université, France.
Courtois
Sophie
S
Block
Jean-Claude
JC
Merlin
Christophe
C
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D019015:Geologic Sediments / Q000737:chemistry* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012722:Sewage / Q000737:chemistry* / Q000382:microbiology
D004269:DNA, Bacterial; D012722:Sewage