Molecular quantification of genes encoding for green-fluorescent proteins.
A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively coamplified DNA standard constructed by point mutation PCR. A single base difference was introduced to achieve a suitable migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant.
Felske
A
A
GBF (German Research Center for Biotechnology), Division of Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. afe@gbf.de
Vandieken
V
V
Pauling
B V
BV
von Canstein
H F
HF
Wagner-Döbler
I
I
D000818:Animals; D004274:DNA, Recombinant / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004783:Environmental Microbiology; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D008628:Mercury / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
D004274:DNA, Recombinant; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008628:Mercury