Quantification of genetically tagged cyanobacteria in Baltic Sea sediment by competitive PCR.
Competitive PCR (cPCR) is a quantitative PCR approach based on the addition of an internal standard to the PCR mixture. In this study, cPCR was used to quantitate genetically tagged cyanobacteria in Baltic Sea sediment. The cyanobacterium Synechocystis 6803-luc has a chromosomal insertion of the firefly luciferase gene, luc, as a marker detectable by PCR. A competitive standard was constructed that contained a 37-bp insertion within the luc DNA region that could be distinguished from the target luc DNA on the basis of size. Synechocystis 6803-luc cells were added to sediment, total bacterial DNA was extracted from sediment and the number of luc DNA copies was quantified by cPCR and phosphor imaging analysis. The internal standard was added either before (co-extraction) or after (post-extraction) isolation of DNA from sediment. The co-extraction method was found to be both more accurate and more precise for the quantitation of added cell numbers (luc DNA copies) by cPCR. When using the co-extraction approach, it was possible to overcome variations in extraction efficiency between replicate samples. The target:competitor PCR product ratio was the only value required for interpolation from standard curves to the luc DNA concentration present in the original sediment sample. The number of luc-tagged cyanobacteria added to the sediment could be calculated after adjustment for the number of chromosomes per cell. This technique should be applicable for quantitation of genetically tagged cyanobacteria in nature.
Möller
A
A
Stockholm University, Sweden.
Jansson
J K
JK
D001345:Autoradiography; D001454:Baltic States; D000458:Cyanobacteria / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D018628:Gene Dosage; D005819:Genetic Markers; D019015:Geologic Sediments / Q000032:analysis / Q000382:microbiology*; D008156:Luciferases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards
D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers; D008156:Luciferases