A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.
Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.
Porcher
C
C
Laboratoire de Génétique Moléculaire, Faculté Xavier Bichat, Paris.
Malinge
M C
MC
Picat
C
C
Grandchamp
B
B
D001331:Automation; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics; D051562:Proto-Oncogene Proteins c-bcr; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr