Extraction, detection and persistence of extracellular DNA in forest litter microcosms.
A DNA extraction method was developed that preferentially extracted extracellular DNA rather than intracellular DNA from forest litter. The method purposely avoided the use of harsh chemicals and physical disruption steps used in total DNA extraction to release DNA from cells. The detection limit of PCR, determined by spiking forest litter samples with a dilution series of Choristoneura fumiferana MNPVegt(-)/lacZ(+) genomic DNA, was about 1 ng DNA or 6.85 x 10(6) target copies 0.5 g(-1) moist forest litter or 0.14 g(-1) dry forest litter. In this study, outdoor terrestrial microcosms, each spiked with 49.2 microg of genomic DNA (from the baculovirus CfMNPVegt(-)/lacZ(+)), were exposed to summer conditions. A 530 bp DNA fragment from the genome of the baculovirus CfMNPVegt(-)/lacZ(+) was detected in these microcosms for about 3 months. The DNA may have persisted for a longer period but was below the detection limit of the PCR analysis.
Laboratory of Microbial Technology, Department of Environmental Biology, University of Guelph, Room 3220, Bovey Building, Guelph, Ont., Canada N1G 2W1.
L S
LS
England
M L
ML
Vincent
J T
JT
Trevors
S B
SB
Holmes
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D004777:Environment; D007313:Insecta / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D014197:Trees / Q000502:physiology*
D004247:DNA