DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types.
With the objective of monitoring xenobiotic degrading bacteria in soil, a method for rapid extraction of DNA from soil, amenable to amplification by PCR, was developed. The method was based on lysis by freeze-thawing and subsequent addition of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide and proteinase K. The extraction method required 2 h and was tested on six different soils differing in organic content, water holding capacity and pH, including ones from which DNA extraction is difficult. DNA yields from the soils ranged from 6.1 to 54.0 micrograms of DNA per g soil. The efficiency and reproducibility of the DNA extraction method were evaluated by competitive PCR. The organic content in the soils was a major factor affecting the amount of obtained DNA amenable for amplification by PCR. A PCR primer-pair was designed on the basis of the known nucleotide sequences of several catechol 2,3-dioxygenase genes. The specificity of the primer-pair was demonstrated on different sequenced catechol 2,3-dioxygenase genes and on site-specific bacterial isolates from polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The concentration of catechol 2,3-dioxygenase DNA in PAH-contaminated sediment undergoing an ex situ compost process was quantified by competitive PCR over a period of 16 weeks. The concentration of PAHs and catechol 2,3-dioxygenase DNA in the soil samples, was found to correlate.
Wikström
P
P
Department of Microbiology, Defence Research Establishment, Umeå, Sweden.
Wiklund
A
A
Andersson
A C
AC
Forsman
M
M
D050557:Catechol 2,3-Dioxygenase; D004269:DNA, Bacterial / Q000302:isolation & purification*; D049308:Dioxygenases; D005069:Evaluation Studies as Topic; D010105:Oxygenases / Q000032:analysis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D004269:DNA, Bacterial; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase