DNA from Oragene®/saliva samples† is compatiblewith TaqMan® SNP genotyping
J. Chartier and H.C. Birnboim

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

DNA collected with the Oragene® self-collection kitworks well with TaqMan® SNP genotyping assays.
SNPs in the thymidylate synthetase and apolipoprotein
E genes were reliably detected.

Introduction
Single-nucleotide polymorphisms (SNPs) are abundant,
and are estimated to occur at 1 out of every 1,000 basesin the human genome1. In addition to diagnostic
applications, SNPs are useful as markers in population
genetics and evolutionary studies2. The TaqMan 5’
nuclease assay is a widely-used SNP genotyping
technology from Applied Biosystems. The purposeof this study was to evaluate the compatibility ofOragene self-collection kits with TaqMan SNP
genotyping assays.

The reaction components for the allelic discrimination
reactions were set up according to Table 2. Although
it is recommended to use 1 to 20 ng of DNA perreaction5, 1 μL of DNA was added to each 25 μL PCR
reaction without adjusting the concentrations. SNP
genotyping reactions were performed on a Rotor-Gene
3000™ real-time quantitative thermal cycler (CorbettResearch) using the cycling conditions in Table 3.
GeneTaqMan Assay ID

Thymidylate
synthetase(TYMS)

Ref SNP ID

C/G C___1637541_1_ rs2298581

Apolipoprotein A/G C___3084818_10
E (APOE)

rs760136

Table 1: TaqMan probes and primers.

Materials and methods
DNA collection
Saliva was collected from 25 donors using Oragene kits.
DNA was purified from 200 μL aliquots of Oragene/saliva samples using the prepIT™•L2P protocol3.
Purified DNA was redissolved in 200 μL of TE buffer(10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA wasquantified using a fluorimeter and SYBR® Green I dye(Molecular Probes) according to the F/D Protocol4.

SNP genotyping
Validated TaqMan SNP genotyping assays wereobtained from Applied Biosystems. The assays aredescribed in Table 1. The probes were labeled with
FAM or VIC dye at the 5’ end, and a minor-groove
binder and non-fluorescent quencher at the 3’ end.

Component

Volume(µL)

2× TaqMan Universal PCR Master Mix

12.5

20× TaqMan SNP Genotyping Assay Mix

1.25

DNA (varying concentrations)

1.0

H2O

10.25

Total volume

25.0

Table 2: Reaction components.

StepTemperature

TimeCycles

195ºC

10 minutes

1292ºC

15 seconds

45

360ºC

1 minute

45

Table 3: Cycling conditions.

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

(outside the USA)(in the USA)

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Results

A/AA/GDNA quantification

Allelic discrimination plots
Figure 1 shows the allelic discrimination plot for thethymidylate synthetase assay and Figure 2 shows
the plot for the apolipoprotein E assay. The plots arepresented as the signal (average fluorescence betweencycles 41 and 45) minus the background (averagefluorescence between cycles 21 and 25).
C/C

Allele C (VIC)

105G/G

042G/G

00

0.5

1.0

1.5

2.0

Allele G (FAM)
Figure 2: Allelic discrimination plot for the apolipoprotein E assay.

TaqMan assays are widely used for SNP genotyping.
As with other molecular genetic techniques, thequality and purity of DNA is important for reliable
results. Our findings indicate that DNA samples
collected with Oragene kits and purified using
prepIT•L2P are suitable for allelic discrimination
using TaqMan probes. Clear discrimination between
different genotypes is evident in Figures 1 and 2.

15

24Discussion and conclusions

G/C

06
Allele A (VIC)

The purified DNA from 25 samples had a medianconcentration of 19.1 ng/μL and ranged from 1.2 to53.4 ng/μL. Consequently, a number of the genotyping
reactions used more than the 20 ng of DNA templaterecommended by the manufacturer.

6Allele G (FAM)
Figure 1: Allelic discrimination plot for the thymidylate
synthetase assay.

Although it is recommended to use 1 to 20 ng of
DNA per reaction, this study used DNA amounts
ranging from 1.2 to 53.4 ng and all 25 samples
gave clear, interpretable results. It was possible toperform the reactions without first determining
the amount of DNA. Thus, the step of adjusting
the DNA concentrations could be avoided.
In summary, DNA collected with Oragene issuitable for SNP genotyping with TaqMan assays.

References
12345Sachidanandam, R., et al. (2001). A map of human genome sequence variation containing 1.42 million single nucleotide
polymorphisms. Nature. 409, 928–933.
Schork, N. J., Cardon, L. R. and Xu, X. (1998). The future of genetic epidemiology. Trends Genet. 14, 266–272.
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 amicro-plate reader. DNA Genotek. PD-PR-075.
June 2004. TaqMan SNP genotyping assays – protocol. Applied Biosystems. Part number 4332856, Rev. B.

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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-011 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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