DNA from Oragene®/saliva samples† and REPLI-g®
whole genome amplification
J. Chartier and M. Pinard

DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

DNA collected with the Oragene® self-collection kti is
successfully amplified using the Qiagen REPLI-g® kit.
10 ng of starting template generates about 40 µg of
amplified DNA for downstream experiments.

redissolved in 75 μL of TE buffer (10 mM Tris-HCl,
1 mM EDTA, pH 8.0). DNA was quantified using
a fluorimeter and SYBR® Green I dye (MolecularProbes) according to the F/D assay7.

Introduction

Whole genome amplification

The Oragene self-collection kit facilitates thecollection and preservation of large amounts of DNA– a median of 110 µg of DNA from 2 mL of saliva1.
Yet a renewable source of DNA may be desirable,
given the rare and valuable nature of many specimens.
Towards this end, Whole Genome Amplification(WGA) allows in vitro production of virtuallyunlimited copies of genomic DNA.

Whole genome amplification of the DNA purified
from Oragene/saliva samples was performed
according to instructions provided with the REPLI-gkit. In brief, 2.5 μL of DNA was added to 2.5 μL ofBuffer D1 (containing KOH & EDTA) to denature theDNA. The sample was incubated for 3 minutes atroom temperature, before adding 5 μL of Buffer N1(neutralizing buffer). Then, 40 μL of Master Mix
containing the REPLI-g DNA polymerase were added
to 10 μL of the denatured DNA solution. The mixture
was incubated overnight at 30°C. After amplification,
the REPLI-g DNA polymerase was heat-inactivatedduring a 10 minute incubation step at 65°C. Table 1summarizes the steps in the protocol.

Multiple Displacement Amplification (MDA) is a
WGA method based on rolling circle amplification
using Phi29 DNA polymerase and random primers.
MDA generates high-fidelity genomic copies withminimal bias2. MDA-amplified DNA has beenvalidated with diverse applications such as SNP
genotyping, STR analysis, comparative genomic
hybridization, RFLP analysis, subcloning, and DNA
sequencing3, 4, 5.
The REPLI-g kit from Qiagen utilizes MDA toexponentially amplify genomic DNA. The purposeof this study was to determine if DNA collected withOragene could be amplified with REPLI-g, and totest the quality of the amplified DNA for PCR andSNP genotyping.

Materials and methods
DNA collection
Saliva was collected from 10 donors using Oragene
self-collection kits. DNA was purified from500 μL aliquots of the Oragene/saliva samples using
the prepIT™•L2P protocol6. Purified DNA was†

The amplified DNA was quantified using the F/D assay.
The molecular weight was analyzed by agarose gelelectrophoresis with ethidium bromide staining andby comparison with a Lambda-Hind III digest ladder.
StepDescription

1Mix 2.5 μL of DNA with 2.5 μL of Buffer D1

2

Incubate at room temperature for 3 minutes3

Add 5 μL of Buffer N1

4Mix with 40 μL of Master Mix

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

6Heat-inactivate sample at 65°C for 10 minutes

7

Store at 4°C or -20°C

Table 1: Summary of the REPLI-g protocol.

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

(outside the USA)(in the USA)

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Polymerase chain reaction

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345WGA and unamplified DNA samples were analyzed
by PCR and agarose gel electrophoresis for a 560 bp
fragment of the human thymidylate synthetase gene.

23.1 kb

SNP genotyping
A TaqMan® SNP Genotyping Assay for thethymidylate synthetase (TYMS) gene was obtained
from Applied Biosystems (TaqMan Assay ID:C___1637541_1_). WGA and unamplified DNA
samples were genotyped using a Rotor-Gene 3000™
real-time quantitative thermal cycler (CorbettResearch). Reactions were set up according to
the manufacturer’s instructions8, 9.

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

Results
Whole genome amplification

1234

For the WGA reactions, the amount of starting DNA
template was 10 ng, and the average amount ofDNA after amplification was 50 ± 1.6 μg, representing
about a 5,000-fold increase. Similarly, 10 ng of thesupplied control DNA was amplified to 58.5 μg.
Figure 1 compares the molecular weight of WGA
and unamplified DNA samples.

Polymerase chain reaction
Figure 2 shows a typical PCR result for WGA andunamplified DNA samples.

SNP genoytping
Figure 3 shows an allelic discrimination plot for thethymidylate synthetase assay. The plot is presented
as the signal (average fluorescence between cycles 41and 45) minus the background (average fluorescence
between cycles 21 and 25).

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 WGA and unamplified DNA, respectively.
 OrageneOragene + REPLI-g

Figure 3: Allelic discrimination plot for the thymidylate synthetase assay.

MK-AN-016 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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2Discussion and conclusions
According to the manufacturer, 10 ng of starting
DNA typically generates 40 µg of high molecularweight amplified DNA10. DNA from Oragene/saliva
samples was successfully amplified with the REPLI-g
kit and gave an average yield of 50 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 µg from2 mL of saliva1. REPLI-g allows DNA from Oragene/saliva samples to be amplified around 5,000-fold.
Additionally, Oragene/saliva samples have been shown
to be stable for extended periods of time at room
temperature11, thereby supplying a long-term sourceof genomic DNA template for WGA reactions.
In summary, DNA collected with Oragene kits
works well with the REPLI-g WGA kit and has thepotential to generate renewable quantities of DNA
for downstream experiments.

References
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Birnboim, H.C. (2004). DNA yield with an Oragene self-collection kit. DNA Genotek. PD-WP-001.
Lasken, R. and Egholm, M. (2003). Whole genome amplification: abundant supplies of DNA from precious samples or clinicalspecimens. Trends Biotech. 21(12): 531-535.
Dean, F. B., et al. (2002). Comprehensive human genome amplification using multiple displacement amplification. PNAS. 99(8): 5261-6.
Hosono, S., et al. (2003). Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13(5): 954-64.
Lage, J. M., et al. (2003). Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement
amplification and array-CGH. Genome Res. 13(2): 294-307.
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 dye
and a micro-plate reader. DNA Genotek, PD-PR-075.
(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.
(January 2005). REPLI-g® handbook. Qiagen.
Birnboim, H.C. (2004). Long-term stability of DNA from saliva samples stored in the Oragene self-collection kit. DNA Genotek. PD-WP-005.

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-016 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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