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Evaluation of Three Parameters for Assessing DNA Quantitation

NCJ Number
Date Published
October 2010
10 pages
This study assessed commercially available DNA quantitation standards, which pertain to a quality assurance step that will ensure there is sufficient DNA in a forensic sample to yield optimal results based on the specifications noted for each amplification kit used.
The study used only one chemistry kit, but the standards were changed in the course of the experiment; they were evaluated by assessing the slope, R2 value, Y-intercept, and the resulting quantities of a serial dilution. Of these three assessed parameters, the accuracy of quantitation most strongly correlated to the Y-intercept, meaning that the Y-intercept is the best indicator of the accuracy of the quantitation result. In addition, the study found that an increase of one point of the CT value on the Y-intercept, 28 to 29 for example, correlates to a two-fold increase in the estimated quantitation value. In general, the study found that Applied Biosystems quantitation standards showed the greatest variability over time and between lot numbers. Another finding was that Promega’s Plexor HY standards showed the least amount of variability over time. In addition, NIST SRM 2372 standards, although accurate, may lose accuracy over a long period of time if stored in a diluted state. In conducting the study, stains were made on Whatman stain cards with 20 ml of a blood sample. After drying the stains, they were packaged and frozen until ready for use. When assessing the quantitation standards, the dried blood-stains were extracted using the BioRobot EZ1 Workstation Trace Tip Dance Protocol and diluted. For each quantitation, a new dried blood sample was used in order to limit the variability in quantitation that may occur due to freezing and thawing of DNA sample extracts. 3 figures and 5 references

Date Published: October 1, 2010