Compatibility of the AUTOPURE® with the Oragene® self-collection kit†
M. Dols‡ J. Chartier** and P. Lem**‡ Gentra Systems, Minneapolis, Minnesota, USA** DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

DNA samples collected with the Oragene® self-collection
kit and purified with the Autopure® LS instrument
have an average DNA yield of 82.3 µg and A260/A280
ratio of 1.87. The DNA is of high molecular weightand suitable for downstream applications like PCR.

Introduction
To maximize the participation rate in population
genetic studies, alternatives to invasive whole bloodcollection are increasing. One such alternative is theOragene self-collection kit (DNA Genotek, Canada).
After saliva is collected, samples can be stored for years
at ambient temperature prior to DNA purification.
Along with the benefit of a non‑invasive collection
method, samples can be shipped at ambient
temperature which saves expensive delivery costs
associated with blood collection. The purpose ofthis study was to assess the yield and quality of
DNA purified on Gentra Systems’ AUTOPURE LS
instrument from the entire 4 mL sample containedin the Oragene kit.

Materials and methods
Sample collection and handling
Saliva samples (2 mL) were collected from 16 individual
donors by following the collection instructions
provided with the Oragene kit. As the kit was capped,
saliva automatically mixed with the 2 mL Oragene
preserving solution. Samples were then incubated
at 50°C overnight in an air incubator and stored atroom temperature. Prior to DNA purification, the4 mL lysate samples were transferred to AUTOPURE
input QUBES and 1 mL of PUREGENE Cell Lysis
Solution containing RNase A was added. Samples
were mixed well by vortexing vigorously.

†Protocol overviewThe lysate samples were placed on the AUTOPURE LS
and the DNA was purified in a fully automatedformat using PUREGENE chemistries (GentraSystems, Inc., Mpls., MN) and the protocol for
1-5 mL cell lysate samples. After samples were loadedon the instrument, Protein Precipitation Solution
was added and the samples were vigorously mixed
to precipitate the proteins. The precipitated proteins
and other contaminants were then collectedby centrifugation and the cleared lysates weretransferred to the output QUBES containing 100%
Isopropanol. The samples were gently mixed andcentrifuged to pellet the precipitated DNA. Residualsalt was removed by washing the samples with
70% Ethanol followed by centrifugation to collectthe DNA pellet. The samples were then hydrated
by adding 300 µL of DNA Hydration Solution
and incubating at 65°C for 1 hour followed byan overnight incubation at room temperature.

UV absorbance determination
DNA yields were determined by UV spectrophotometry
using a Beckman DU-64 spectrophotometer(Beckman-Coulter, Fullerton, CA). A 10 µL volume ofeach DNA sample was diluted with 190 µL ultrapure
deionized water and vortexed at high speed for
5 seconds. Absorbencies were measured at 260 nm,
280 nm, and 320 nm (background). To calculate
DNA concentration, the 320 nm background reading
was subtracted from the 260 nm reading, and theresulting value was multiplied by the DNA extinction
coefficient of 50 µg/mL and the dilution factor(200 µL/10 µL). The DNA yield was calculated bymultiplying each concentration by the respective
DNA volume. The A260/A280 ratio was determinedby subtracting the background reading from eachof the two readings before they were divided.

Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

Some DNA Genotek products may not be available in all geographic regions, contact your sales representative for details.

www.dnagenotek.com • support@dnagenotek.com

Genomic DNA analysis
To assess DNA integrity, 100 ng from each sample
was separated by 0.7% agarose gel electrophoresis.
The DNA was electrophoresed for 1 hour at 75 volts
using 0.125 µg/mL of ethidium bromide in the gel.
The gel was photographed on a UV transilluminatorusing a Kodak Digital Imaging System EDAS 120 LE(Rochester, NY). Lambda DNA digested with HindIII
was used as a size reference.

DNA amplification
Purified DNA was evaluated for amplification
performance using primers specific to a 1.5 kb target
in one of the cytochrome P450 genes (CYP2D6 locus).
For each reaction, 100 ng of DNA was amplified in
a 50 µL volume containing: 1×Epicentre Failsafe™
Buffer E (Madison, WI), 1.0 µM of Primer MP1and MP2 (Genosys, The Woodlands, TX), 2.5 Uof Promega Taq polymerase (Madison, WI).
The amplification conditions for the CYP2D6 targetwere: 30 cycles: 94°C for 30 seconds, 63°C for
30 seconds, and 72°C for 2 minutes; 72°C holdfor 6 minutes, 4°C hold. A volume of 10 µL fromeach reaction was analyzed by 1% agarose gelelectrophoresis. The DNA was electrophoresedfor 60 minutes at 75 V using 0.125 µg/mL ethidium
bromide in the gel and running buffer. A 1 kb
ladder was used as a size reference. The gel wasphotographed on a UV transilluminator usinga Kodak Digital Imaging System EDAS 120 LE(Rochester, NY).

SampleDNA yield (µg)

A260/A280

11121.83245.8

1.85

3267

1.81

4

36.7

1.91

5

153

1.84

6101

1.87

7

62.4

1.79

8

45.2

1.89

9

18.4

1.99

1060.6

1.84

1132.3

1.90

12

21.0

1.99

13102

1.81

14

29.1

1.93

15119

1.84

16111

1.84

Average

82.3

1.87

Table 1: DNA Yield and quality of saliva collected with Oragene kits
and purified on the AUTOPURE LS instrument with PUREGENE chemistries.
DNA was purified from 16 individual samples following the 1-5 mL
cell lysate protocol on the instrument. The DNA was analyzed byUV spectrophotometry to determine DNA yield and A260/A280 ratios.

Results
DNA yields and A260/A280 readings from the samples
purified on the AUTOPURE LS Instrument are
shown in Table 1. The average DNA yield fromthe Oragene/saliva samples was 82.3 µg of DNA.
The average A260/A280 was 1.87, which suggests
pure DNA free of contaminants. Electrophoretic
analysis of the purified DNA samples (Figure 1)
shows high quality DNA was purified for all samples,
as indicated by the high molecular weight band of
at least 50 kb and the lack of low molecular weightDNA. The purified DNA amplified well using
the primer set for the CYP2D6 locus (Figure 2).

MK-AN-005 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Lane

Description

1, 20

Uncut Lambda

2, 19

Lambda x HindIII Ladder

3-18

Samples 1-16

Figure 1: Integrity of DNA purified from Oragene/saliva samples on
the AUTOPURE LS instrument. DNA was purified from 16 individual
samples following the 1-5 mL cell lysate protocol on the instrument
and analyzed by agarose gel electrophoresis to determine the qualityof the DNA.

www.dnagenotek.com • support@dnagenotek.com

2

Lane

Description

1, 18

1 kb ladder

2-17

Samples 1-16

Figure 2: Amplification of DNA purified from Oragene/saliva samples
on the AUTOPURE LS instrument. DNA was purified from 16 individual
samples following the 1-5 mL cell lysate protocol on the instrument
and subsequently amplified using primers for the CYP2D6 locus.
Amplified DNA was analyzed by agarose gel electrophoresis.

DNA was excellent. In addition, a 1.5 kb target wasamplified successfully from the purified DNA samples,indicating the DNA is free of contaminants or inhibitorsand suitable for PCR analysis. The AUTOPURE
instrument purifies high yields of high quality DNA
in a fully automated high-throughput format,
processing up to 128 samples of 4 mL Oragene/saliva
samples during an 8 hour shift.

Recommendations
To purify from the entire 4 mL saliva sample collectedwith Oragene, add 1 mL of Cell Lysis Solution to theinput QUBES along with the 4 mL lysate sample andfollow the AUTOPURE LS 1.0-5.0 cell lysate protocol.

Discussion and conclusions
The AUTOPURE LS Instrument purifies DNA in afully automated format from the entire saliva sample
collected in Oragene, producing high quality DNA
that is free from detectable protein contamination.
Agarose gel electrophoresis analysis of the purified
samples showed that the integrity and quality of theOragene®•DNA is not available for sale in the United States.
Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
®Oragene is a registered trademark of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

MK-AN-005 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

3