Degradable dU-based DNA template as a standard in real-time PCR quantitation.
Development of real-time quantitative PCR assays requires suitable positive controls. For assays with clinical applications, these controls may be difficult to obtain because some molecular aberrations are rare and patient material may be available in limited amounts. Because of the risk of introducing contaminations in the laboratory, cloned DNA is not a desirable alternative. We describe the use of dU-containing DNA as a positive control template in real-time quantitative PCR. dU-DNA constructs can be decontaminated by adding uracil N-glycosylase (UNG) to the reaction mixture. In addition, dU-DNA can be used for accurate quantification, because it allows quantification to be expressed in numbers of molecules. Since synthetic dU-DNA constructs can easily be quantitated spectroscopically, they provide a more accurate control than arbitrary cell line units. We applied this method for the detection of the E2A-Pbx1 gene fusion and show that UNG-containing reactions can be employed for diagnostics without loss of sensitivity, and that for positive and quantitative controls UNG negative reactions can be used. The use of dU-DNA provides a novel type of control template that can easily be integrated into existing PCR protocols.
Pennings
J L
JL
Central Hematology Laboratory, University Medical Center St Radboud Nijmegen, Nijmegen, The Netherlands.
Van De Locht
L T
LT
Jansen
J H
JH
Van der Reijden
B A
BA
De Witte
T
T
Mensink
E J
EJ
D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000592:standards*; D045647:DNA Glycosylases; D003856:Deoxyuracil Nucleotides / Q000032:analysis / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D051981:Uracil-DNA Glycosidase
D003856:Deoxyuracil Nucleotides; D018398:Homeodomain Proteins; D015514:Oncogene Proteins, Fusion; C089847:E2A-Pbx1 fusion protein; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase