Compatibility of the PUREGENE® DNA purification kit
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 Puregene® have an average
DNA yield of 85.5 µg and A260/A280 ratio of 1.79.
The DNA is high molecular weight and is suitablefor 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 withblood collection. The purpose of this study was to assess
the yield and quality of DNA purified manually with
Gentra Systems’ PUREGENE chemistries from theentire 4 mL sample contained in the Oragene kit.

Materials and methodsSample set-up
Oragene kits were used to collect 2 mL saliva samples
from 16 individual donors following the instructions
for collection provided with the kits. Samples werethen incubated at 50°C overnight in an air incubatorand stored at room temperature. Prior to DNA
purification, the 4 mL lysate samples were transferred
to 50 mL tubes and 1 mL of PUREGENE Cell Lysis
Solution containing 25 µL RNase A was added. Samples
were mixed well by vortexing vigorously. The DNA
was purified following protocol 400244 for 4 mLsaliva samples collected in Oragene kits. The samples
were hydrated by adding 300 µL of DNA Hydration
Solution and incubating at 65°C for 1 hour, followedby an overnight incubation at room temperature.
†UV absorbance determination
DNA yields were determined by UV spectrophotometry
using a Beckman DU-64 spectrophotometer (BeckmanCoulter, Fullerton, CA). A 10 µL volume of each DNA
sample was diluted with 190 µL ultrapure deionizedwater and vortexed at high speed for 5 seconds.
Absorbencies were measured at 260 nm, 280 nm, and320 nm (background). To calculate DNA concentration,
the 320 nm background reading was subtracted from the260 nm reading, and the resulting value was multipliedby the DNA extinction coefficient of 50 µg/mL and thedilution factor (200 µL/10 µL).
The DNA yield was calculated by multiplying eachconcentration by the respective DNA volume. The
A260/A280 ratio was determined by subtracting thebackground reading from each of the two readings
before they were divided.

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 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 a50 µL volume containing: 1×Epicentre Failsafe™ Buffer E(Madison, WI), 1.0 µM of Primer MP1 and MP2(Genosys, The Woodlands, TX), 2.5 U of Promega

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

(outside the USA)(in the USA)

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Taq polymerase (Madison, WI). The amplification
conditions for the CYP2D6 target were: 30 cycles:
94°C for 30 seconds, 63°C for 30 seconds, and 72°C
for 2 minutes; 72°C hold for 6 minutes, 4°C hold.
A volume of 10 µL from each reaction was analyzed
by 1% agarose gel electrophoresis. The DNA waselectrophoresed for 60 minutes at 75 V using 0.125 µg/mLethidium bromide in the gel and running buffer. A
1 kb ladder was used as a size reference. The gel was
photographed on a UV transilluminator using a Kodak
Digital Imaging System EDAS 120 LE (Rochester, NY).

Results
DNA yields and A260/A280 readings from thesamples purified manually with PUREGENE are
shown in Table 1. The average DNA yield from thecollected Oragene/saliva samples was 85.5 µg of DNA.
The average A260/A280 was 1.79, which suggests pureDNA free of contaminants. Electrophoretic analysisof the purified DNA samples (Figure 1) showshigh quality DNA was purified for all samples, asindicated by the high molecular weight band ofat 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).
Sample
12
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Average

DNA yield (µg)
107
61.1
200
63.8
147
112
76.6
60.1
31.5
49.7
39.1
37.5
130
39.2
111
103
85.5

A260/A280
1.771.78
1.79
1.78
1.78
1.83
1.73
1.79
1.80
1.80
1.76
1.81
1.78
1.84
1.79
1.82
1.79

Lane
1, 20
2, 193-18

Description
Uncut Lambda
Lambda x HindIII Ladder
Samples 1-16

Figure 1: Integrity of DNA manually purified using PUREGENE fromOragene/saliva samples. DNA was purified from 16 individual samples
following the PUREGENE 4 mL saliva/Oragene protocol and analyzed
by agarose gel electrophoresis to determine the quality of the DNA.

Lane
1, 182-17

Description
1 kb ladder
Samples 1-16

Figure 2: PCR amplification of DNA manually purified using
PUREGENE from Oragene/saliva samples. DNA was purified from
16 individual samples following the PUREGENE 4 mL saliva/Oragene
protocol and subsequently amplified using primers for the CYP2D6locus. Amplified DNA product was analyzed using agarose gelelectrophoresis.

Discussion and conclusions
High yields of high quality DNA were purified fromOragene/saliva samples using the PUREGENE DNA
purification kit. Agarose gel electrophoresis analysisof the purified samples showed that the integrityand quality of the DNA was excellent. In addition,a 1.5 kb target was amplified successfully fromthe purified DNA samples, indicating the DNA isfree of contaminants or inhibitors, and suitable for
PCR analysis.

Table 1: DNA yield and quality of saliva collected with Oragene
and purified manually using PUREGENE. DNA was purified from
16 individual samples following the PUREGENE 4 mL saliva Oragene
lysate protocol. The DNA was analyzed by UV spectrophotometry
to determine DNA yield and A260/A280 ratios.
Oragene®•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-003 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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