Quantification of Genetically Tagged Cyanobacteria in Baltic Sea Sediment by Competitive PCR

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Abstract
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 (postextraction) 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 coextraction approac...

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