Kiranmai Durvasula1, Jeff Roeder1, Travis Butts1, Mike Farrell1.
1Omega Bio-tek, Norcross, GA
With advances in genomics research and clinical diagnostics, there is a need for rapid, high throughput and cost-effective technologies for purifying high-quality DNA from cheek swab samples. DNA Genotek's ORAcollect (Figure 1) offers a painless, non-invasive method for collection of oral cavity DNA samples. DNA from these samples has been extracted using Omega Bio-tek's Mag-Bind® Blood and Tissue DNA HDQ 96 Kit (M6399). Here, we demonstrate a streamlined workflow for processing up to 96 ORAcollect oral samples in a single run on two open ended automation platforms, the Hamilton Microlab® STAR and Qiagen Biosprint® 96.
Figure 1. ORAcollect (OC-100) Collection Kit for human oral samples.
Cheek swab samples were collected from 8 independent donors into ORAcollect tubes. These tubes come prefilled with a proprietary bacteriostatic reagent which not only inhibits the growth of bacteria, but also lyses and preserves the sample DNA. The DNA isolation from these samples was then carried out using the Mag-Bind® Blood and Tissue DNA HDQ 96 Kit from Omega Bio-tek, skipping the lysis step. The Mag-Bind® HDQ beads provide quick magnetic response and allow for automation. The workflow for DNA extraction using Hamilton and Qiagen platforms is outlined in Figure 2.
Figure 2. Schematic of the DNA extraction workflow.
The workflow with Qiagen involved a few manual steps (~2) whereas with Hamilton, it was totally hands off. Both the workflows were comparable in terms of overall protocol time (~55-65 minutes). Also the ORAcollect tubes contain barcodes that can be decoded by most commercially available scanners and can be integrated into the Hamilton workflow, ensuring full sample traceability throughout the process.
DNA yield extracted on Hamilton and Qiagen was quantified using Promega QuantiFluor® dsDNA system. To test for the integrity of the purified DNA and its direct suitability for downstream applications, real-time PCR was performed on triplicates of undiluted, 10-fold and 100-fold diluted DNA extracts. Agilent's Brilliant III 2X SYBR® mix and universal human primers were used following a standard amplification protocol on the ABI 7900.
Figure 3. DNA yield.
DNA yield from ORAcollect samples when extracted on Hamilton Microlab® STAR and Qiagen Biosprint® 96 are shown in Figure 3.
Ct values of the undiluted samples extracted on either of the automation platforms are as listed in Table 1 and they indicate positive amplification. There was no detectable fluorescence in the no template control wells. Typically, Ct of the samples whose concentration differs by a factor of 10 are ~3.3 cycles apart. The average ΔCt between the 10-fold and undiluted samples was lower than 3.3 for both Hamilton and Qiagen, indicative of presence of inhibitors. As expected, the average ΔCt values at higher dilutions (100-fold and 10-fold) were 3.21 and 3.31 for the same, indicating good PCR efficiency.
|Sample||Hamilton Microlab® STAR||Qiagen Biosprint® 96|
|1||21.85 ± 0.13||21.25 ± 0.09|
|2||23.60 ± 0.11||22.84 ± 0.08|
|3||22.58 ± 0.09||20.57 ± 0.36|
|4||23.62 ± 0.14||24.30 ± 0.12|
|5||22.16 ± 0.15||20.53 ± 0.08|
|6||21.42 ± 0.19||20.37 ± 0.23|
|7||21.40 ± 0.51||21.05 ± 0.56|
|8||20.40 ± 0.18||20.08 ± 0.33|
Table 1. Ct values of undiluted DNA extractions run in triplicate.
A rapid, reliable and high throughput solution for extracting DNA from ORAcollect samples, in conjunction with Omega Bio-tek's extraction chemistry, was ported onto two open ended automation platforms, Hamilton Microlab® STAR and Qiagen Biosprint® 96. The DNA obtained was of sufficiently high quality and suitable for a variety of downstream applications.
|DNA Genotek||OC-100||ORAcollect 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)|