Human genomic DNA content of saliva samples collectedwith the Oragene® self-collection kit†
C. James, R.M. Iwasiow, H.C. Birnboim
DNA Genotek, Ottawa, Ontario, Canada

Estimating the amount of human genomic DNA
in oral samples is important because normal oral
samples (as compared to blood samples) containvariable amounts of bacterial DNA. We determinedthat the median bacterial DNA content in a seriesof 50 Oragene®/saliva samples was 11.8% of the total
DNA, suggesting that 88.2% was of human origin. As
an independent measure of the human DNA content,
the TaqMan® RNase P assay was used. When corrected
for efficiency using bacteria-free blood DNA from thesame donors, the human DNA content was determined
to be 80%. Thus, two complementary methods allow
the same conclusion, that the vast majority of DNA
in Oragene/saliva samples is of human origin.

Introduction
For any type of genetic analysis (e.g., clinical genetictesting, paternity testing, pharmacogenomic testing,population studies), non-invasive methods andself-collection techniques are preferred. For thisreason, Oragene/saliva samples are often used as aconvenient source of high quality DNA. Though
Oragene/saliva samples contain a small amount ofbacterial DNA in addition to human genomic DNA,
this bacterial DNA has minimal practical significance.
Numerous studies have shown that DNA from
Oragene/saliva samples gives equivalent results toDNA from blood for applications such as PCR, SNP
genotyping, microarrays and next-generation
sequencing1, 2, 3. Furthermore, of all oral collection
methods (e.g., swabs, mouthwash, cytobrush), theOragene kit provides the highest yield and highest
quality of human genomic DNA4, with the lowest
bacterial DNA content. This technical bulletin
provides quantitative information about the amountof human genomic DNA in Oragene/saliva samples,
as estimated by two different methods.

†Materials and methods
Sample collection
Paired blood and saliva samples were collected from50 donors. After providing informed consent, eachdonor was first asked to deliver 2 mL of saliva into
an Oragene kit according to the instructions foruse included with the kit. The saliva collection wasunsupervised. Shortly after collection, the Oragene/saliva samples were mixed by shaking for 15 seconds
and then stored at room temperature. A phlebotomist
then drew 8 mL of venous blood from each donor
into a BD Vacutainer EDTA tube (BD Cat. No. 36643).
The blood samples were immediately placed onto arocker and allowed to mix for >10 minutes to avoid
the formation of microclots.

DNA purification
Saliva: several days after collection, a 500 µL aliquotof saliva was purified according to the optimized
Oragene purification protocol using prepIT™•L2P(DNA Genotek) for 0.5 mL5.
Blood: immediately after collection, the buffy coat
was prepared by spinning the collected samples at
2500 × g for 10 minutes; plasma was discarded andthe buffy coat was transferred to a microcentrifuge
tube. DNA was purified from the buffy coat using
Qiagen’s QIAamp blood mini kit (Cat. No. 51106)
according to the manufacturer’s instructions.
Purified DNA from both sources was stored at -20°C.

DNA quantification
Aliquots of purified DNA from both blood andsaliva were thawed at room temperature and thenincubated at 50°C for 30 minutes to ensure that thehigh molecular weight DNA was fully dissolved.

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

(outside the USA)(in the USA)

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The total DNA concentration was determined byfluorescence using SYBR® Green I dye (Invitrogen,Cat. No. S-7563) and a Rotor-Gene™ 6000 real-timethermal cycler (Corbett Life Science) according to theDNA Genotek DNA Quantification assay protocol6.
The DNA was then diluted in TE to 7.5 ng/µL andthe final concentration was verified using the DNAQuantification assay.
TaqMan RNase P assay to measure human genomic
DNA: a Standard Curve was prepared using Human
Genomic DNA supplied with the TaqMan RNase PDetection Reagents. A standard curve was preparedby serial dilution (5 ng to 30 ng/reaction).
Real-time PCR: the human content of the bloodand saliva DNA samples was measured directly using
the TaqMan™ RNase P Detection reagents (FAM)(ABI, Cat. No. 4316831) and the TaqMan Universal
PCR mastermix no AMPErase UNG (ABI, Cat. No.
4324018). Real-time PCR was done according to themanufacturer’s specifications. 15 ng (2 µL of 7.5 ng/µL)of total DNA was added to each reaction and thepercent of human content was determined by dividing
the amount of human DNA (as determined by theTaqMan assay) by the total amount of DNA added
to the reaction (as determined by the DNA
Quantification assay).

amount of bacterial DNA (as determined by theBacterial DNA assay) by the total amount of DNA
added to each reaction (as determined by theDNA Quantification assay).

Results
Comparison of DNA samples spiked and not spiked
with E. coli control DNA showed that the real-time
PCR reaction was not 100% efficient; without thecorrection, the true amount of bacterial DNA would
be underestimated. The median percent of bacterial
DNA content, as determined by the Bacterial DNA
assay, of total DNA from 50 blood samples was 0.03%(range 0% to 0.48%). The median percent of bacterial
content of total DNA purified from 50 saliva samples
was 11.8% (range 2.0% to 39.9%) (Table 1, Figure 1).

Bacterial DNA assay
The bacterial DNA assay developed by DNA Genotek
was employed. The detailed protocol is available
upon request.

Figure 1: Bacterial DNA as a percentage of total DNA in 50 Oragene/saliva or blood samples. The horizontal line represents the median value.

Standard Curve: purified E. coli control DNA wasobtained from Sigma (E. coli, strain B, Cat. No. D4889).
A standard curve was prepared using a serial dilution(0.31 ng to 20 ng/reaction).
Real-time PCR: PCR primers were chosen from a
region of the 16S rRNA gene that is known to beconserved across a wide variety of microorganisms,
and is not found in eukaryotes7. The blood and saliva
DNA was tested for the presence of 16S rRNA geneby real-time quantitative PCR using a Rotor-Gene
6000 real-time thermal cycler. Each reaction used
15 ng (2 µL of 7.5 ng/µL) total DNA, and the efficiencyof the reactions was determined by running a second
15 ng aliquot of sample spiked with 5 ng of E. coli
control DNA in parallel. The percent bacterial DNA
content was determined by dividing the corrected

PD-WP-011 Issue 4/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 2: Human DNA as a percent of total DNA in 50 Oragene/saliva
and blood samples. Left panel summarizes the TaqMan assay results
while the right panel displays data normalized to 100% human DNA
in blood samples. The horizontal lines represent the median value.

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% Bacteria
Donor
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

Blood
0.04%
0.04%
0.03%
0.03%
0.04%
0.03%
0.04%
0.04%
0.03%
0.03%
0.17%
0.48%
0.06%
0.31%
0.12%
0.45%
0.12%
0.09%
0.12%
0.09%
0.05%
0.06%
0.04%
0.06%
0.02%
0.02%
0.02%
0.03%
0.08%
0.09%
0.01%
0.01%
0.00%
0.01%
0.01%
0.00%
0.01%
0.01%
0.01%
0.01%

Saliva22.04%
5.53%
11.47%
9.47%
13.01%
3.16%
21.16%
10.70%
14.39%
2.83%
4.52%
9.91%
2.29%
14.12%
15.84%
15.10%
23.36%
12.03%
18.70%
18.04%
29.79%
17.63%
6.72%
7.46%
10.44%
12.77%
3.94%
8.79%
3.52%
22.84%
12.41%
8.08%
4.60%
39.86%
12.30%
2.04%
2.72%
23.66%
23.13%
14.98%% HumanBlood
109.62%98.73%
88.74%
108.80%93.39%
88.69%
105.53%100.46%100.19%102.41%75.36%
82.50%
68.81%
82.95%
70.58%
76.48%
97.17%
97.58%
82.96%
94.03%
67.26%
82.90%
65.66%
71.44%
81.31%
73.64%
65.68%
76.75%
77.15%
74.83%
73.37%
88.46%
87.03%
88.30%
87.49%
84.79%
79.57%
86.57%
68.40%
78.83%

Saliva61.48%
70.69%
71.51%
85.86%
66.25%
85.24%
66.44%
102.31%76.49%
84.32%
64.28%
56.43%
55.55%
41.39%
50.15%
46.57%
63.32%
81.16%65.84%
74.82%
60.34%
73.98%
62.07%
60.71%
79.72%
63.99%
69.95%
57.81%
13.33%
53.67%
68.72%
73.60%
73.48%
51.27%
66.36%
68.31%
66.76%
55.85%
58.03%
66.69%PD-WP-011 Issue 4/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

% Bacteria
Donor
41
42
43
44
45
46
47
48
49
50
Median

Blood
0.02%
0.02%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.03%

Saliva18.58%
7.09%
6.54%
3.72%
13.94%
15.66%
4.74%
22.55%
4.84%
5.98%
11.75%% HumanBlood
101.22%87.66%
67.52%
77.14%
73.31%
78.14%
69.40%
97.33%
85.70%
74.53%
82.92%

Saliva74.99%
56.54%
65.83%
90.11%
70.60%
66.59%
76.59%
58.62%
37.44%
43.53%
66.30%

Table 1: Detailed results for each donor showing the calculated
bacterial or human DNA as a percent of total DNA from Oragene/saliva or blood samples.

Discussion
Some previous studies have reported that the amountof human genomic DNA in oral samples is only asmall percentage of the total DNA due to the presenceof a large amount of bacterial DNA. For example,
Feigelson et al. (2001) found that the medianpercentage of bacterial DNA in mouthwash samples
was 66% of the total DNA.
Similarly, Garcia-Closas et al. (2001) found thatthe median percentage of bacterial DNA was 50.5%
in mouthwash samples and 88.5% in cytobrushsamples (Table 2).
Collection
methodMedian percentageof bacterial DNA

References

Mouthwash

66.0%

Feigelson et al. (2001)

Mouthwash

50.5%

Garcia-Closas et al. (2001)

Cytobrush

88.5%

Garcia-Closas et al. (2001)

Oragene/saliva

11.8%

Table 2: Comparison of the median percentage of bacterial DNA
obtained using various oral collection techniques.

In contrast to these reports, we report that thevast majority of DNA from Oragene/saliva samples
is of human origin, with a median bacterial content
of only 11.8%. The difference between our results
and reports using other collection methods can beexplained by the fact that the Oragene kit contains

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antibacterial agents, which prevent the growth ofbacteria between the time of collection and the timeof DNA purification.
In this report, we have used two complementarymethods to assess the amount of human DNA present
in the total DNA purified from saliva samples.
First, we used a Bacterial DNA assay developed byDNA Genotek, which directly measures bacterial
DNA content using real-time PCR targeting a highly
conserved region of bacterial 16S rRNA gene. Our
assay includes an internal standard to more accurately
calculate the amount of bacterial DNA in a sample.
PCR efficiency was decreased by an average of 25.9%
when analyzing both blood and saliva samples,suggesting it was independent of the purification
method. After correction for PCR efficiency, theBacterial assay indicated that DNA from blood had
a median bacterial content of 0.03%, and DNA fromsaliva had a median bacterial content of 11.8%. This
low amount in blood is expected since blood fromhealthy individuals contains no bacteria. In contrast,
there are bacteria in the oral cavity and therefore saliva
can be expected to contain bacterial DNA. Notably,bacterial DNA in saliva samples collected in the Oragene
kit is appreciably lower than oral samples collectedby other methods (Table 2). The results of the Bacterial
DNA assay indicate that a median of ~12% of oral
DNA collected with Oragene kit is bacterial andsuggest that a median of 88% is of human origin.
To complement the results of the Bacterial DNA assay,
we used a second method (the TaqMan RNase P
Detection assay) to estimate the content of human

DNA in saliva. As a control, the same assay wasused to measure the human DNA content of blood,
which is 100% human in origin. Presumably due toincomplete removal of all inhibitors during routine
isolation of DNA from blood, the human DNA
content was only 82.9% (range 65.7% to 109.6%)(Figure 2 and Table 1). If one were to assume that theremainder was bacterial DNA, one would incorrectly
conclude that the bacterial content was 17.1%,
something that clearly cannot be the case for a blood
sample. Similarly, the percentage of human DNA
from saliva is underestimated to be 66.3% (Figure 2and Table 1), an amount that corresponds to previously
published results using this assay8. By normalizing
the 82.9% value obtained for blood DNA to 100%,
then the true value for human DNA in saliva canbe estimated to be 80%, a value in good agreementwith the Bacterial DNA assay results.

Conclusion
In summary, our Bacterial DNA assay (when usedwith an internal standard) is as accurate a method
as currently available for estimating the amount ofbacterial DNA in a sample. This method estimates
the human DNA in saliva samples, collected with theOragene kit, to be about 88%. An independent method
using the TaqMan RNase P assay produced similar
results. We, therefore, conclude that the Oragene
saliva sample kit is a reliable method for obtaining
large amounts of human genomic DNA containing
significantly less bacterial contamination than otheroral collection methods.

References
12345678Ahituv, N., Kavaslar, N., Schackwitz, W., Ustaszewska, A., Collier, J.M., Hebert, S., Doelle, H., Dent, R., Pennacchio, L.A. andMcPherson, R. (2006). A PYY Q62P variant linked to human obesity. Human Molecular Genetics. 15, 387-391.
Reynolds, J., Do, T., Hongo, D., Kuramoto, K., Biggs III., W. and Frech, C. (2007). Comparison of high density genotyping resultsfrom saliva and blood samples on Affymetrix GeneChip® GenomeWide SNP 6.0 arrays.
Iwasiow R.M. and Tayeb M. (2011). Evaluation of pharmacogenetic markers by exome-sequencing of DNA extracted fromsaliva samples. DNA Genotek. MK-00048.
Rogers, N., Cole, S., Lan, H., Crossa, A. and Demerath, E. (2007). New saliva DNA collection method compared to buccal cellcollection techniques for epidemiological studies, American Journal of Human Biology. 19, 319-326.
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 anda micro-plate reader. DNA Genotek. PD-PR-075.
Muyzer, G., de Waal, E. and Uitterlinden, A. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis
analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied Environmental Microbiology. 59, 695-700.
Rylander-Rudqvist T., Håkansson N., Tybring G. and Wolk A. (2006). Quality and quantity of saliva DNA obtained from theself‑administrated Oragene method – a pilot study on the cohort of Swedish men. Cancer Epidemiol Biomarkers Prev. 9, 1742-1745.

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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.

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