SPECIFIC QUANTITATION OF GENOMIC DNA IN THE FEMTOGRAM RANGE BY AMPLIFICATION OF REPETITIVE SEQUENCES

An assay was developed to specifically quantitate concentrations as lo w as 50 fg/ml genomic DNA based on the amplification of repetitive seq uences. Reliable results were obtained by using internal standard mole cules which were coextracted, coamplified, and coanalyzed with the nuc leic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers foll owed by quantitation of fluorescence derived hom 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 rea ction products, the primary results of the assay were obtained as copi es per milliliter of sample. These were converted into mass units of D NA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA and the sequences amp lified 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. (C) 1996 Academic Press, Inc.