Validation of reduced-scale reactions for the Quantifiler Human DNA kit.
Accurate quantification of DNA samples is an important step in obtaining accurate and reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved the speed and accuracy of DNA quantification over earlier methods, albeit at significantly greater cost per reaction. Here, the performance of reduced volume (10 microL) DNA quantification assays using the Quantifiler Human DNA Quantification Kit was evaluated using commercial standards and single source biological stains (e.g., venous blood, saliva, and semen). In addition, casework-type samples including those subjected to environmental contaminants containing PCR inhibitors and samples having undergone extensive DNA degradation were also quantified. The concentration of DNA in various forensic samples ranged from 0 to 2.9 ng/microL depending on sample source and/or environmental insult. Compared to full-scale reactions, reduced volume assays displayed equivalent to improved amplification efficiency and sample-to-sample reproducibility (+/-0.01-0.17 C(T FAM)). Furthermore, the use of data from reduced-scale Quantifiler reactions facilitated the accurate determination of the amount of sample DNA extract needed to generate quality STR profiles. The use of 10 microL-scale Quantifiler reaction volumes has the practical benefit of increasing the effective number of reactions per kit by 250%; thereby reducing the cost per assay by 60% while consuming less sample. This is particularly advantageous in cases of consumptive testing.
Department of Biological Sciences, University of Denver, 2190 E. Iliff Ave., Denver, CO 80208, USA. cwestrin@du.edu
Christian G
CG
Westring
Richard
R
Kristinsson
Dustin M
DM
Gilbert
Phillip B
PB
Danielson
D001769:Blood; D004247:DNA / Q000302:isolation & purification*; D053836:DNA Degradation, Necrotic; D016172:DNA Fingerprinting / Q000295:instrumentation*; D005838:Genotype; D006197:Hair; D006801:Humans; D008297:Male; D009061:Mouth Mucosa; D016133:Polymerase Chain Reaction; D012463:Saliva; D012661:Semen; D012987:Soil; D020080:Tandem Repeat Sequences
D012987:Soil; D004247:DNA