Overcoming bacterial DNA contamination in real-time PCR and RT-PCR reactions for LacZ detection in cell therapy monitoring.
Since the introduction of the polymerase chain reaction the presence of contaminating bacterial DNA in the Taq polymerase preparations has hampered the use of this technique in microbiology. Lately, this inconvenience has equally impeded gene quantification in the field of cell or gene therapy, where bacterial genes such as LacZ are often used as tags to detect vectors or cells after their injection in the recipient organism. Several means to overcome the DNA contamination of Taq Polymerase have been reported with variable degrees of decontamination efficiency and alteration of the PCR reaction. Here we propose two protocols to efficiently quantify DNA or RNA from the LacZ gene by real-time PCR using either decontamination by low concentrations of DNAse I prior to PCR amplification or a highly purified Taq Polymerase which is devoid of detectable contamination.
Tondeur
Sylvie
S
Inserm E0-03, Institut Universitaire d'Hématologie, AP-HP, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France.
Agbulut
Onnik
O
Menot
Marie-Laurence
ML
Larghero
Jérôme
J
Paulin
Denise
D
Menasche
Philippe
P
Samuel
Jane-Lise
JL
Chomienne
Christine
C
Cassinat
Bruno
B
D000818:Animals; D001854:Bone Marrow Cells; D017690:Cell Transplantation / Q000592:standards*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D003850:Deoxyribonuclease I; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000592:standards*; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019914:Taq Polymerase / Q000302:isolation & purification
D004269:DNA, Bacterial; D019914:Taq Polymerase; D003850:Deoxyribonuclease I