Specific quantitation of genomic DNA in the femtogram range by amplification of repetitive sequences.
An assay was developed to specifically quantitate concentrations as low as 50 fg/ml genomic DNA based on the amplification of repetitive sequences. Reliable results were obtained by using internal standard molecules which were coextracted, coamplified, and coanalyzed with the nucleic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers followed by quantitation of fluorescence derived from the reaction products after separation by PAGE. Based on the known amount of added internal standard molecules and the intensities of the fluorescence of the reaction products, the primary results of the assay were obtained as copies per milliliter of sample. These were converted into mass units of DNA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA, and the sequences amplified were CR1 repetitive elements. This type of assay may be applied in cases where a sensitive and precise quantitation of genomic DNA is required, such as in the quality control of biological products.
Himmelspach
M
M
Biomedical Research Center, Immuno AG, Orth/Donau, Austria.
Gruber
F
F
Antoine
G
G
Falkner
F G
FG
Dorner
F
F
Hämmerle
T
T
D000818:Animals; D001483:Base Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000706:statistics & numerical data; D002645:Chickens; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes; D005784:Gene Amplification; D016678:Genome; D008832:Microchemistry / Q000379:methods / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA