Kiranmai Durvasula1, Jeff Roeder1, Travis Butts1, Bassam El-Fahmawi2.
1Omega Bio-tek, Inc. 2MAWI DNA Technologies
High quality DNA is a critical factor determining success of many molecular biology applications, such as next generation sequencing (NGS), qPCR, microarrays, etc. Other potential applications include clinical molecular diagnostics, genetic analyses, pharmacogenomics, animal speciation studies, and forensics. Buccal swabs or saliva are the most commonly used sample types for these kinds of analyses because of their non-invasiveness and ease of collection. However, these existing non-invasive technologies suffer from low genomic DNA recovery along with high bacterial contamination. Current swab-based products require manual swab removal and are not automation-friendly. MAWI DNA Technologies' iSWAB collection device addresses these issues with its innovative tube insert design that maximizes the release of cells collected on swabs into an existing proprietary selection mammalian cell lysis buffer and allows for long term, room temperature storage of the sample while preventing the release of bacterial DNA (Figure 1). The design is particularly attractive because it eliminates the need for swab retention and is compatible with a variety of of commercially available swabs (Figure 1). LIMS compatible barcodes are also included on each iSWAB device for efficient sample traceability and storage purposes.
Omega Bio-tek offers the Mag-Bind® Blood and Tissue DNA HDQ 96 Kit for rapid and reliable isolation of high quality DNA from blood samples, saliva, swabs, mouse tails, dried blood spots and cultured cells, which is capable of being automated on open-ended liquid handling platforms such as the Hamilton Microlab® STAR. The Omega/Hamilton automated protocol is capable of extracting DNA from 96 samples in under an hour. Here, we provide a comprehensive, automated workflow for the extraction of gDNA from iSWAB oral samples utilizing Omega Bio-tek's extraction chemistry on the Hamilton Microlab® STAR.
Twenty-one independent oral samples were collected in iSWAB collection devices, 9 of which where collected with Copan FLOQSwabs™ and the rest with Puritan cotton swabs. The samples were sent to our laboratory by 2-day ground shipping and stored at room temperature for 14 days from the time of collection to the time of DNA purification. The automation protocol began with 250 µL of the samples from the iSWAB collection devices being rapidly arrayed into 96 wells using 300 µL Hamilton Slim CO-RE® tips (Figure 2). Hamilton Microlab® STAR™ was programmed to perform various liquid handling and magnetic bead-based tasks as demanded by the Mag-Bind® Blood and Tissue DNA HDQ 96 protocol for the extraction of genomic DNA. The extracted DNA was quantified using Promega QuantiFluor® dsDNA system.
Performance Evaluation of the Purified DNA
Purifed DNA from 5 samples collected using Puritan cotton swabs were selected for the proof-of-concept study. The quality of purified DNA for downstream applications was assessed by real-time PCR performed on triplicates of 1X (~12 ng), 10X, 100X, 1,000X and 10,000X serial dilutions. Agilent's Brilliant 2X SYBR® mix and universal human primers were used following a standard amplification protocol on the ABI 7900. The real-time PCR performance was evaluated based on qPCR reaction efficiency and R2 value.
Bacterial DNA Estimation
The assay as described in  was performed to quantify the percentage bacterial contamination in the samples. We chose 5 of the Copan FLOQ™ collected samples as our data set for this assay. Briefly, real-time was performed with the PCR primers from the 16S rRNA gene that is known to be conserved across a wide variety of prokaryotic microorganisms. Serial dilutions of pure bacterial DNA (here, E. coli) was used to construct a standard curve, which was later used to estimate the amount of bacterial DNA in the iSWAB samples. A known quantity of E. coli (5 ng) was included as our internal standard (IS) to more accurately calculate the bacterial DNA in the sample. Each PCR reaction used 14 ng of DNA sample (TD). To compensate for variations in amplification efficiency between the two runs, two aliquots of the same sample was run in parallel - one spiked with IS (S) and the other unspiked (US). A correction factor (CF) was calculated as the ratio of IS to the difference between the spiked and unspiked. The percent bacterial DNA contamination in the sample compensating for the differences in PCR reaction efficiency was estimated as below:
% Bacterial DNA = CF x US/TD x 100
The purified DNA yield from the iSWAB samples are shown in Figure 3. Standard curves were generated by 5-fold serial dilutions of purified DNA in the range of 0.0012 ng to 12 ng. Figure 3 depicts a representative image of the standard curve. 10,000X dilution was omitted from the curve as the Ct values were close to that of the no template controls and no longer accurate. Linear regression was performed on the data points to arrive at the slope and regression co-efficient (R2). The slope of the standard curve was employed to determine the qPCR efficiency (E = 10-1/slope - 1) x 100).
Table 1 lists the real-time PCR efficiencies and R2 values for each of the 5 samples tested. A robust linear correlation was observed (R2 > 0.99) for all the 5 samples tested. qPCR efficiency ranged from 86.56% to 96.16%. We observed a good fidelity in the Ct values among the 4 replicates tested at each dilution with the average standard deviation ranging from 0.17 to 0.55 depending on the sample (Table 1). The above discussed parameters suggest that the isolated DNA was of high quality and suitable for downstream applications.
|Puritan Cotton Sample||qPCR Efficiency||R2||Average Standard Deviation|
|Copan FLOQ Sample #||% Bacterial Contamination|
DNA from oral samples typically contain bacterial DNA in addition to human genomic DNA. This bacterial DNA has no practical relevance and hinders performance of applications downstream. Hence, we estimated the bacterial DNA as a percent of total DNA in the iSWAB samples as described in . Our findings indicate bacterial DNA ranged from 0.15 to 4.50% of the total DNA yield among the samples tested (Table 2). A median of 2.34% of the total oral DNA collected with MAWI's iSWAB collection device was found to be bacterial. In comparison, bacterial DNA content with the iSWAB device is must lower than various existing oral collection techniques (Table 3).
|Collection Method||Median % of Bacterial DNA||Reference|
An automated, high throughput protocol was developed for rapidly extracting DNA from MAWI's iSWAB collection devices utilizing Omega Bio-tek's extraction chemistry on the Hamilton Microlab® STAR™. The process yielded high quality, amplifiable DNA suitable for a variety of downstream applications. MAWI's iSWAB collection device offers a reliable option for oral sampling with significantly less bacterial contamination when compared with other techniques. It has potential to make significant contributions, especially in the areas of human genome analyses, forensics, and applications such as NGS where bacterial contamination poses a serious problem.
|MAWI DNA Technologies||iSWAB-DNA-1200||iSWAB DNA Collection Kit|
|Omega Bio-tek||M6399-00||Mag-Bind® Blood and Tissue DNA HDQ 96 Kit (1 x 96 preps)|
|M6399-01||Mag-Bind® Blood and Tissue DNA HDQ 96 Kit (4 x 96 preps)|
. DNA Genotek, "Bacterial DNA Assay," PD-PR-065 Issue 4, 2012.
. DNA Genotek, "Human Genomic DNA Content of Saliva Samples Collected in Oragene® DNA," PD-WP-011 Issue 4, 2012.
*Microlab®, STAR™ and CO-RE® are trademarks of the Hamilton Company in the U.S. and other countries.