Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.
Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.
Koponen
Jonna K
JK
A. I. Virtanen Institute, University of Kuopio, Kuopio, FIN-7021, Finland.
Turunen
Anna-Mari
AM
Ylä-Herttuala
Seppo
S
D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli; D007202:Indicators and Reagents; D007763:Lac Operon / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D019914:Taq Polymerase / Q000032:analysis* / Q000235:genetics
D004269:DNA, Bacterial; D007202:Indicators and Reagents; D019914:Taq Polymerase