This study evaluated the performance of reduced-volume (10 ml) DNA quantification assays with the use of the Quantifiler Human DNA Quantification Kit, using commercial standards and single-source biological stains (e.g., venous blood, saliva, and semen), as well as casework-type samples that included those subjected to environmental contaminants and samples with extensive DNA degradation.
The study demonstrated the same performance between the 25 ml and 10 ml quantifiler reaction volumes. This reduction in reaction volume has the practical benefit of increasing the effective number of reactions per kit by 250 percent. This reduces the cost per assay by 60 percent while consuming less sample. The amplification efficiency and reproducibility of reduced-scale reactions were found to be virtually indistinguishable or to trend slightly better than the manufacturer's recommended protocol for amplification of a broad range of dilutions of the commercially prepared human DNA standard provided with the Quantifiler kit. The same equivalence in performance was achieved with single-source blood samples extracted from cotton swabs. With more challenging mixtures of blood and top soil, the reduced-scale reactions produced equivalent to slightly more precise quantification results than full-scale assays of the same DNA extract. Reduced-scale reactions were used to quantify the DNA content of a variety of simulated casework samples and postmortem blood samples that contained highly degraded DNA. The description of materials and methods addresses DNA sample preparation and extraction, quantitative real-time PCR, and STR genotyping. 3 tables, 7 figures, and 29 references