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This journal is © The Royal Society of Chemistry 2017 Analytical Methods, 2017, XX, XX | 1

Supplementary Information: Enabling Transport of Genomic DNA through Porous Membranes for Point-of-Care Applications

Figure S1. Adsorption of DNA in porous membranes. Heat-fragmented S. aureus DNA was diluted in buffer (pH 5, 6.5, or 8.5) and added directly to 1×1 cm membranes. Samples sat at room temperature for 5 minutes followed by fluid collection by centrifugation at 10,000 ×g for 3 minutes. Elution volumes were measured and the total amount of recovered DNA was quantified by qPCR. Recoveries were adjusted for elution volume: % recovery = [qPCR output (copies/µL) * elution volume (µL)] / input (copies). Averages of at least N=4 are reported with error bars representing standard error of the mean. Buffers containing no DNA (“Blank samples”) were also added to and eluted from the membranes to test for any effects on qPCR. The Blank samples eluted from the nitrocellulose membranes showed slight enhancement of qPCR signals which may explain both the variability and the observed recovery for DNA from nitrocellulose.

Electronic Supplementary Material (ESI) for Analytical Methods.This journal is © The Royal Society of Chemistry 2017

ARTICLE Analytical Methods

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Figure S2. Effect of proteinase K (PK) treatment on samples post lysis and thermal fragmentation. All samples, expect S. aureus, were lysed by heating to 95°C for either 2 or 8 minutes; S. aureus was treated with ACP for 2 minutes at room temperature prior to heating to 95°C. For all samples and heating times tested, treatment with PK does not improve flow through the porous membrane suggesting that proteins in the sample are not significantly contributing to DNA movement. Averages of N=3 are reported with error bars representing standard error. [p-values for transported comparing heat only v. PK treatment]. A. N. gonorrhoeae [2 min: 0.073; 8 min: 0.079], B. S. aureus [2 min: 0.112; 8 min: 0.079], C. E. coli [2 min: 0.338; 8 min: 0.085], and D. HeLa epithelial cells [2 min: 0.820; 8 min: 0.073].

Figure S3. Lysis efficiencies for various methods and heating times at 95°C. Treatment with ACP + 10 minutes of heating was set to 100% (dashed red line) for each pathogen tested. In general, lysis efficiencies were similar for each condition. Samples treated with ACP could not have a 0 min heat condition because the non-denatured enzyme inhibits qPCR. The average of N=6 (N=3 from two independent cultures) is reported with the error bars representing standard error. A. E. coli treated with and without ACP, and B. MRSA treated with ACP.

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Figure S4. Example PFGE for varying E. coli heating time at 95°C. E. coli containing samples were treated with ACP at room temperature for 2 minutes followed by heating to 95°C for 0-10 minutes. A qualitative assessment of fragmentation was visualized using PFGE with N=3 samples per heating time. As heating time increased, the amount of DNA retained in the wells decreased as did the amount of longer fragments.

Figure S5. Extended E. coli heating times at 95°C. Increased heating times to 20–25 minutes results in a higher percentage of DNA that transports through the membrane indicating a higher degree of fragmentation. The slight drop-off at 30 minutes may be caused by DNA degradation. Averages of N=3 are reported with error bars representing standard error.

ARTICLE Analytical Methods

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Figure S6. Lysis efficiencies for various methods and heating temperatures for 10 minutes. Treatment with ACP + 10 minutes of heating at 95°C was set to 100% (dashed red line) for each pathogen tested. In general, lysis efficiencies were similar for each condition across temperatures for each pathogen. Heat only lysis was less effective than heat + ACP for E. coli. The average of N=6 (N=3 from two independent cultures) is reported with the error bars representing standard error. A. E. coli treated with and without ACP, and B. S. aureus treated with ACP.

Figure S7. Example PFGE for varying E. coli heating temperatures for 10 minutes. E. coli

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containing samples were treated with ACP at room temperature for 2 minutes followed by heating to 85–95°C for 10 minutes. A qualitative assessment of fragmentation was visualized using PFGE with N=3 samples per heating time. As heating temperature increased, the amount of DNA retained in the wells decreased as did the amount of longer fragments.