Visual Automated Fluorescence Electrophoresis Provides Simultaneous Quality, Quantity, and Molecular Weight Spectra for Genomic DNA from Archived Neonatal.

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Visual Automated Fluorescence Electrophoresis Provides Simultaneous Quality, Quantity, and Molecular Weight Spectra for Genomic DNA from Archived Neonatal Blood Spots Tara L. Klassen, Janice Drabek, Torjbörn Tomson, Olafur Sveinsson, Ulrika von Döbeln, Jeffrey L. Noebels, Alicia M. Goldman The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 283-290 (May 2013) DOI: 10.1016/j.jmoldx.2013.01.003 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Extraction of gDNA and WGA from decades-old 3-mm blood spots assessed for quality, quantity, and molecular-weight spectra. A: The flowchart defines our current QC paradigm (gray) to assess sample quality, quantity, and integrity of blood spot–derived gDNA samples compared with VAFE (black). The dotted black line shows the ability of VAFE to assess all QC parameters simultaneously even after the initial extraction, enabling efficient sample characterization for entry into downstream genomic applications. B: Quantification of total gDNA extracted from a single 3-mm blood spot using the QiaAmp Mini Kit measured using 260/280 ratio of a Nanodrop (black bar) and the VAFE on the TapeStation (gray bar). The 260/280 ratio overestimates dsDNA, whereas VAFE uses a fluorescent Sybr dye to calculate concentration, which is only capable of intercalating in dsDNA, resulting in a more accurate quantification. C: VAFE quantification of gDNA extracted from a single 3-mm blood spot before (gray bar) and after (dark bar) WGA. The WGA yields approximately 1 μg of dsDNA from a starting template of approximately 10 ng. D: Slab gel analysis of gDNA integrity reveals that pre-WGA DNA is not detectable on the slab gel, whereas all post-WGA samples are of high molecular weight comparable to reference DNA samples (275 and 12.5 ng) extracted from venous blood. The extracted gDNA spot (Q) is run in parallel with the post-WGA amplification (W) of the same template. E: Visualization of pre- and post-WGA blood spot samples using VAFE shows high-molecular-weight DNA is extracted from the decades-old blood spot. This high-molecular-weight DNA (>48,500 bp) is enriched in the post-WGA sample. F: Fluorescent electropherograms of the DNA extracted from a blood spot collected in 1975 and 1982. The pre-WGA DNA (black) contains high-molecular-weight (7000 to 48,500 bp) DNA. In both blood spots the post-WGA (gray) sample shows an enrichment of heavy fragments, but the relative amount of small to large fragments differs greatly between the samples, suggesting that samples must be evaluated for quality on an individual basis. The Journal of Molecular Diagnostics 2013 15, 283-290DOI: (10.1016/j.jmoldx.2013.01.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions