DNA from Oragene®/saliva samples† and whole genome amplification
with GenomiPhi™
J. Chartier
DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

DNA collected with the Oragene® self-collection kit
is successfully amplified using the GenomiPhi™ DNA
amplification kit and has the potential to generate
near-unlimited quantities of DNA for downstream
experiments.

Introduction
As the genotyping of single-nucleotide
polymorphisms (SNPs) becomes more automatedand less costly, the limiting factor for genome-wide
genetic analysis is the amount of DNA that can becollected from study subjects. A renewable source ofgenomic DNA is desirable. The traditional methodof transforming lymphoblastic cell lines is usually too
expensive for population studies with large samplesizes. A less expensive alternative is whole genome
amplification (WGA), which allows in vitro productionof numerous copies of the entire genome.
Multiple displacement amplification (MDA) is aWGA method that uses Phi29 DNA polymerase
and random priming. MDA generates high-fidelity
genomic copies with minimal bias1.
The GenomiPhi DNA amplification kit fromAmersham Biosciences utilizes MDA to exponentially
amplify genomic DNA. The purpose of this study wasto determine if DNA collected with Oragene could be
amplified with GenomiPhi and to test the quality of
the amplified DNA for PCR and SNP genotyping.

Materials and methods
DNA collection
Saliva was collected from 10 donors using Oragene
self-collection kits. DNA was purified from200 μL aliquots of Oragene/saliva samples using
the prepIT™•L2P protocol2. Purified DNA was†redissolved in 200 μL of TE buffer (10 mM Tris-HCl,
1 mM EDTA, pH 8.0). DNA was quantified using afluorimeter and SYBR® Green I dye (Molecular Probes)
according to the F/D protocol3.

Whole genome amplification
Following instructions provided with the GenomiPhikit, 1 μL of DNA was added to 9 μL of sample buffer
containing random hexamers and heated to 95°C todenature the DNA. The sample was cooled and mixed
with 9 μL of reaction buffer containing salts anddeoxynucleotides and 1 μL of enzyme mix. The mixture
was incubated overnight at 30°C. After amplification,
the Phi29 DNA polymerase was heat-inactivatedduring a 10 minute incubation at 65°C. Table 1
summarizes the steps in the protocol.
The amplified DNA was quantified using the F/Dprotocol. The molecular weight was analyzed byagarose gel electrophoresis with ethidium bromide
staining and comparison with a Lambda-Hind III
digest ladder.
StepDescription1

Mix 1 μL of DNA with 9 μL of sample buffer

2

Heat to 95°C for 3 minutes

3

Cool to 4°C on ice

4

Mix cooled sample with 9 μL of reaction
buffer and 1 μL of enzyme mix

5Incubate sample overnight at 30°C (16-18 h)

6Heat-inactivate sample at 65°C for 10 minutes7Store at 4°C or -20°C

Table 1: Summary of the GenomiPhi protocol.

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.

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Polymerase chain reaction

Polymerase chain reaction

WGA and original unamplified DNA samples wereanalyzed by PCR and agarose gel electrophoresis
for a 560 bp fragment of the human thymidylate
synthetase gene.

Figure 2 shows a typical PCR result for WGA andunamplified DNA samples.
1234

SNP genotyping
A TaqMan SNP Genotyping Assay for the thymidylate
synthetase (TYMS) gene was obtained from Applied
Biosystems (TaqMan Assay ID: C___1637541_1_).
WGA and original DNA samples were genotyped
using a Rotor-Gene 3000™ real-time quantitative
thermal cycler (Corbett Research). Reactions were set
up according to the manufacturer’s instructions4, 5.

Results
Whole genome amplification
For the WGA reactions, the average amount ofstarting DNA template was 13 ± 2 ng (mean ± SEM).
The control DNA supplied with the kit had aconcentration of 10 ng/μL. The average amountof DNA after amplification was 7.60 ± 0.13 μg,representing about a 600-fold increase. Figure 1
compares the molecular weight of WGA andunamplified DNA samples.
1234560 bp

Figure 2: PCR results. Lane 1 shows PCR with unamplified control
DNA and Lane 2 contains a 100 bp ladder. Lanes 3 and 4 show PCR
with amplified and unamplified DNA respectively.

SNP genotyping
Figure 3 shows an allelic discrimination plot for thethymidylate synthetase assay. The plot is presentedas the signal (average fluorescence between cycles 41and 45) minus the background (average fluorescence
between cycles 21 and 25).
Oragene
C/C

G/C

30.0

5Allele C (VIC)

23.1 kb

Oragene + WGA

20.0

10.0
G/G

Figure 1: Agarose gel electrophoresis of DNA samples. Lane 2 contains
unamplified DNA sample. Lanes 3 and 4 show WGA reactions withsample and control DNA respectively. A Lambda-Hind III digest wasused as the marker in Lanes 1 and 5.

0.0
0.0

10.0

20.0

Allele G (FAM)
Figure 3: Allelic discrimination plot for the thymidylate
synthetase assay.

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Discussion and conclusions
According to the manufacturer, 10 ng of starting
DNA should generate 4 to 7 µg of amplified DNA
with a high molecular weight6. DNA from Oragene
was successfully amplified with the GenomiPhi kit
and gave an average yield of 7.60 µg, slightly higher
than the expected amount. The amplified DNA
worked well for both PCR and TaqMan reactions.
The WGA DNA gave similar PCR bands to theoriginal unamplified DNA and the SNP genotyping
calls were identical.

With Oragene, the median DNA yield is 110 µgfrom 2 mL of saliva7. GenomiPhi allows DNA
from Oragene to be amplified around 600-fold.
Furthermore, this amplified DNA may be used as
the template for subsequent WGA reactions. Up to
4 re-amplifications have been performed with noapparent effect on PCR of various loci across thehuman genome8. In summary, DNA collected withOragene works well with the GenomiPhi WGA kit
and has the potential to generate near‑unlimited
quantities of DNA for downstream experiments.

References
12345678Lasken, R. and Egholm, M. (2003). Whole genome amplification: abundant supplies of DNA from precious samples or clinicalspecimens. Trends Biotech. 21(12): 531-535.
Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.
DNA quantification using the Fluorescence/DNase (F/D) assay. Replaced with DNA quantification using SYBR Green I dyeand a micro-plate reader. DNA Genotek. PD-PR-075.
June 2004. TaqMan SNP genotyping assays – protocol. Applied Biosystems. Part number 4332856, Rev. B.
Chartier, J. and Birnboim, H.C. (2005). DNA from Oragene/saliva samples is compatible with TaqMan SNP genotyping.
DNA Genotek. MK‑AN-011.
(2003). GenomiPhi DNA amplification kit – instructions. Amersham Biosciences. 74004229 Ed. AB.
Birnboim, H.C. (2004). DNA yield with an Oragene self-collection kit. DNA Genotek. PD‑WP-001.
(Checked February 2, 2005). GenomiPhi FAQ and Troubleshooting. http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/autodna_genomiphi_faqs#Use.

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 and prepIT™ is a 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-012 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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