Comparative Analytical Utility of DNA Derived from Alternative Human Specimens for Molecular Autopsy and Diagnostics Tara L. Klassen, Eva-Lotta von Rüden,

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Presentation transcript:

Comparative Analytical Utility of DNA Derived from Alternative Human Specimens for Molecular Autopsy and Diagnostics Tara L. Klassen, Eva-Lotta von Rüden, Janice Drabek, Jeffrey L. Noebels, Alica M. Goldman The Journal of Molecular Diagnostics Volume 14, Issue 5, Pages 451-457 (September 2012) DOI: 10.1016/j.jmoldx.2012.04.005 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Comparison of experimental steps inform the development of an efficient universal extraction protocol for ATS samples. A: The flowchart defines which ATS samples were compared in this study, as well as the subsequent wash, extraction, and optional purification steps tested. The heavy black line shows the flow of the universal extraction protocol for Guthrie-based samples, whereas the dotted line represents the workflow for nail-derived DNA. B: Total DNA yield obtained from 3-mm blood spots using three common wash solutions; TE (1× TE), SDS (Tris/0.1% SDS), and FTA (FTA wash solution) in quantitatively similar amounts; however, the addition of detergent produces a spot devoid of colored heme and associate proteins. C: After 1 hour of incubation at 56°C, the high salt–containing extraction buffers (STE, PBS) with ProK had more total DNA yield from blood spots compared with TE/ProK; however, only DNA in TE produces a raw DNA extract compatible with downstream applications without the need for additional DNA purification. D: Total DNA yield increased linearly with increasing number of blood spots in the extraction using the universal extraction protocol. E: The home protocol can reproducibly extract total DNA from Guthrie card–based samples, including all ages of blood spots as well as buccal scrapes. In all panels, the box plots represent both the range (error bars = min/max values) and distribution (dark half = 25% to 50%; light half = 50% to 75%; interface = mean) of total DNA quantified using SYBR Green. The Journal of Molecular Diagnostics 2012 14, 451-457DOI: (10.1016/j.jmoldx.2012.04.005) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions