Non-invasive, assisted collection of high quantity andquality genomic DNA from saliva of young children
J.O. Niles, A. Jackson, S. Rabuka, and R.M. Iwasiow
DNA Genotek, Ottawa, Ontario, Canada

Abstract

Materials and methods

Large population-based studies, involving thousandsof subjects, are increasingly being used to investigate
the genetic determinants of complex diseases. Saliva
is a convenient source of genomic DNA because
it can be collected in a painless and non-invasive
manner. Non-invasive methods and techniques thatpermit self-collection are preferred because they
increase compliance rates and reduce costs. For thisreason, many large-scale studies now use DNA fromsaliva collected using the Oragene® kit† as the sourceof genomic DNA for downstream applications.
Human genomic DNA from saliva can be used forgenotyping, sequencing and micro-array analysis.
In order to facilitate the non-invasive collectionof genomic DNA from a population of all ages(including those individuals who cannot spit) we
describe a new method of using sponges to transfer
saliva from a donor’s mouth into an Oragene kit.
Unlike buccal swabs, this method transfers saliva
which contains high quality and quantity DNA into
the Oragene kit which preserves the DNA and prevents
bacterial growth. We report that this method allows
for the collection of a median yield of 17.3 µg ofgenomic DNA with median A260/A280 ratio of 1.8 anda molecular weight > 23 kb. The collected samples
are stable at ambient temperature for years andcan be used in a multiple downstream applications.

1 Place one sponge in cheek pouch.
Gently move the sponge along thegums and inner cheeks for 30 seconds
to soak up as much saliva as possible.

V-notchFill line

2 Once the sponge is saturated withsaliva, insert sponge in V-notch offunnel. Wring saliva out of sponge usinga twisting and pushing motion against
the inner wall of the V-notch. Saliva
will flow into tube.
3 Repeat these steps (1 to 2) USING THE
SAME SPONGE until the liquid saliva(not bubbles) reaches the fill line.
Check sponge for damage each time
before inserting into donor’s mouth.
Use second sponge if first spongeshows any signs of wear or tear.
Tap tube bottom against hard surface
to reduce number of bubbles.
4 Hold the tube upright with one hand.
Close the lid with the other hand (asshown) by firmly pushing the lid until
you hear a loud click. The liquid in thelid will be released into the tube tomix with the saliva. Make sure the lid
is closed tightly.
5 Hold the tube upright. Unscrew thefunnel from the tube.

6 Use the small cap to close thetube tightly.

7 Shake the capped tube for 5 seconds.
Discard or recycle the funnel.
Discard sponges.

Figure 1: Assisted collection of saliva using a sponge with theOragene kit.

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

(outside the USA)(in the USA)

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Results
7−10 years
17%

0−1 year8%
10 - 15 minutes
9%< 3 minutes
20%

6 -10 minutes
20%

1−3 years39%

5−7 years22%> 15 minutes
6%

3 - 6 minutes
45%

3−5 years
14%

Figure 2: Distribution of donor ages

Figure 3: Time required to collect saliva to the fill-to-line••••

Children aged 9 months to 10 years old
were recruited for this study.
Median age of children was 3.5 years.
Samples from a total of 77 children were analyzed.
90

7060
50
40
30
20
100Figure 4: Total amount of DNA recovered.

••
•
•

220
Concentration of purified DNA (ng/µL)

Total amount of DNA recovered (µg)

8065% of all donors irrespective of age were able
to complete the collection of saliva in under
6 minutes.

200
180
160
140120100
8060
40200Figure 5: Concentration of DNA purified from collected saliva.

Collected samples were purified according to the prepIT™•L2P (DNA Genotek) purification protocol1.
Purified DNA was quantified by fluorescence using SYBR® Green I dye2.
The total amount of DNA collected from each child is reported in Figure 4. The median amountof DNA recovered was 17.3 µg.
The resulting purified DNA had a median concentration of 60.2 ng/µL.

PD-WP-018 Issue 2/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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22.0A260/A280 ratio

1.9
1.8
1.7
1.6

12
3
4
5678910
1112
13
14
15
16
1718
19202122232425
26
27
28
2930
31
32
33
34
35
36

1.5Sample ID
Figure 6: Corrected A260/A280 ratio of purified DNA.

••The corrected A260/A280 ratio was calculated by subtracting the A320 value (which represents
scattered light due to insoluble material) from both the A260 and A280 values.
The purified DNA had a median A320 corrected A260/A280 ratio of 1.82.

Figure 7: Representative agarose gel showing high molecular DNA.

•The molecular weight of purified DNA was assessed by running a 0.8% agarose gel. The purified
DNA consistently had a molecular weight > 23 kb as compared with the Lamda-Hind III ladder.

PD-WP-018 Issue 2/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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10 -0.2
10 -0.4
10 -0.6
10 -0.8

Norm. Fluoro.

10 -1.0
10 -1.2
10 -1.4
10 -1.6 Threshold
10 -1.8
10 -2.0
10 -2.2
10 -2.4
10 -2.6
10 -2.812345678

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 40Cycle
Figure 8: Real-time PCR results of human thymidylate synthetasegene.

••

Purified DNA was suitable for use in real-time PCR. Purified DNA was diluted to 4 ng/µL and 20 ng wasused in the PCR reaction.
Orange lines represent the 20 samples tested. Each sample performed equally well and no inhibition wasobserved. Black line represents the no template control.

Conclusions•••

This study enrolled 77 children ranging in age from 9 months to 10 years. Following the instructions
provided, an adult assisted with the collection of saliva samples from each of the donors. Using a sponge,
saliva was successfully collected from all 77 participants.
Following sample purification using the prepIT purification protocol1, the quality of the sample wasassessed. The analytical specifications of all samples are summarized in Table 1.
This method provides a simple non-invasive technique for collecting large amounts of high quality genomic
DNA suitable for downstream applications.
Age of donor

3.5 years

Total DNA yield

17.3 µg

Purified DNA concentration

60.2 ng/µL

A260/A280 ratio

1.82

Table 1: Summary of study results; median values reported.

References
12

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.

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.

PD-WP-018 Issue 2/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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