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

• Published in: BioTechniques Vol. 22; no. 3; pp. 512 - 518
• Main Authors: Möller, Annelie, Jansson, Janet K
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.03.1997; Eaton
• Subjects: Animal, plant and microbial ecology; Autoradiography; Baltic States; Biological and medical sciences; Biotechnology; Cyanobacteria - genetics; Cyanobacteria - isolation & purification; Diverse techniques; DNA Primers; DNA, Bacterial - analysis; DNA, Bacterial - isolation & purification; Electrophoresis, Agar Gel; Fundamental and applied biological sciences. Psychology; Gene Dosage; General aspects; Genetic engineering; Genetic Markers; Genetic technics; Geologic Sediments - analysis; Geologic Sediments - microbiology; Luciferases - genetics; Methods. Procedures. Technologies; Microbial ecology; Miscellaneous; Molecular and cellular biology; Polymerase Chain Reaction - methods; Reference Standards; Synthetic digonucleotides and genes. Sequencing; Baltic Sea; Baltic States; Recombinant microorganism; Genetic marker; Enzyme; Photinus-luciferin 4-monooxygenase (ATP-hydrolysing); Competitive reaction; Synechocystis; Gene expression; Sediments; Polymerase chain reaction; Cyanobacteria; Bacteria; Oxidoreductases; Detection; Quantitative analysis
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/97223rr02

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 6803- has a chromosomal insertion of the firefly luciferase gene, , as a marker detectable by PCR. A competitive standard was constructed that contained a 37-bp insertion within the DNA region that could be distinguished from the target DNA on the basis of size. 6803- cells were added to sediment, total bacterial DNA was extracted from sediment and the number of 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 ( DNA copies) by cPCR. When using the coextraction 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 DNA concentration present in the original sediment sample. The number of -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.