Genetic material is isolated from individuals for population screening, clinical molecular diagnostics and paternity testing. Collection of human saliva samples is a simple, noninvasive alternative to intravenous blood collection. DNA Genotek’s Oragene•DNA Self Collection Kits offer convenient collection in a solution that stabilizes saliva samples for prolonged storage at room temperature.
The Maxwell® 16 System was developed to meet the needs of low- to moderate-throughput users by providing automated purification at a scale appropriate to their workload without considerable capital investment, training or maintenance
. The DNA purification capabilities of the Maxwell® 16 Instrument made it an ideal complement to the Oragene•DNA/saliva-sample-collection kits. Therefore, we studied the ability of the Maxwell® 16 System to extract DNA from saliva samples collected using Oragene•DNA kits.
Materials and Methods
Oragene•DNA Self-Collection Kits contain 2ml of DNA-preserving solution (DNA Genotek Cat.# OG-250). Two milliliters of saliva was collected from 16 donors (4ml total volume of Oragene•DNA/saliva sample) using the kits. The Oragene•DNA/saliva samples were incubated for 2 hours at 50ºC, as directed by the manufacturer, prior to DNA purification with the Maxwell® 16 System.
DNA was extracted from 200µl and 400µl of Oragene•DNA/saliva samples using the Maxwell® 16 Instrument with the Maxwell® 16 Tissue DNA Purification Kit (Cat.# AS1030) and Maxwell® 16 Blood DNA Purification Kit (Cat.# AS1010). For both kits, purified DNA was eluted in the recommended volume of 300µl, following the 35-minute procedure
. For comparison, DNA was also extracted from 200µl and 400µl of Oragene-DNA/saliva samples using the manual Oragene•DNA protocol
Oragene•DNA/saliva samples extracted using the Maxwell® 16 Tissue DNA Purification Kit (Cat.# AS1030) yielded an average of 3.4µg and 5.6µg DNA from 200µl and 400µl samples, respectively, as determined by absorbance measurements (Figure 1). Yields were not significantly different from samples isolated using the Maxwell® 16 Blood DNA Purification Kit (Cat.# AS1010) or the manual Oragene•DNA protocol. Between the two Maxwell® 16 kits, the highest average DNA concentration was achieved using a 400µl sample with the Maxwell® 16 Tissue DNA Purification Kit: 26ng/µl with an A260/A280 purity ratio >1.7.
Figure 1. DNA yield from Oragene•DNA/saliva samples using the Maxwell® 16 System or the manual Oragene•DNA protocol.
Oragene•DNA/saliva samples (200µl or 400µl) were processed using the Maxwell® 16 Tissue DNA Purification Kit (Cat.# AS1030), Maxwell® 16 Blood DNA Purification Kit (Cat.# AS1010) or manual Oragene•DNA method. DNA yield and purity were determined by absorbance measurements at 260nm and 280nm using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific).
DNA extracted by each method was analyzed by agarose gel electrophoresis. Each extraction method yielded DNA of comparable high molecular weight (~23kb; Figure 2). The DNA performance was evaluated in multiplex real-time PCR using the Plexor® HY System (Figure 3). Samples reproducibly amplified and did not show any inhibition of the Plexor® HY internal PCR control (data not shown).
Figure 2. Agarose gel electrophoresis of DNA isolated from 400µl of Oragene•DNA/saliva samples.
Purified genomic DNA was analyzed on a 0.7% agarose gel with ethidium bromide staining. Five microliters of eluate was loaded in each well. Lane M, Lambda DNA/HindIII Marker (Cat.# G1711); lane 1, saliva DNA purified using the Maxwell® 16 Tissue DNA Purification Kit (Cat.# AS1030); lane 2, saliva DNA purified using the Maxwell® 16 Blood DNA Purification Kit (Cat.# AS1010); lane 3, saliva DNA purified using the manual Oragene•DNA protocol.
Figure 3. Real-time PCR of DNA isolated from Oragene•DNA/saliva samples using the Maxwell® 16 System.
DNA samples were analyzed for performance in real-time PCR using the Plexor® HY System (Cat.# DC1000) on the Applied Biosystems 7500 FAST instrument. Two microliters of eluate was analyzed per 20µl amplification reaction. Shown are four samples (green, yellow, red and blue traces) analyzed in replicate and compared to the Plexor® HY Male Genomic DNA Standard (black traces).
The Maxwell® 16 System can purify up to 16 Oragene•DNA/saliva samples in as little as 35 minutes. The DNA is of high purity and high molecular weight, and performs well in downstream applications, such as real-time PCR. For highest yield and concentration, we found the Maxwell® 16 Tissue DNA Purification Kit was best when used with 400µl of saliva sample.