Increasing DNA yield by collecting more saliva
E. Doukhanine and J. Chartier
DNA Genotek Inc, Ontario, CanadaAbstract
The Oragene®•DNA Self-Collection Kit instructs donors tocollect 2 mL of saliva. If more DNA is desired, up to 4 mL ofsaliva can be collected for a proportional increase in DNAyield.

Figure 1 shows the molecular weights of DNA purified
from the 2 and 4 mL saliva samples after storage atroom temperature for 98 days.

Donor

Introduction
The Oragene•DNA Self-Collection Kit collects a medianof 110 μg of DNA from 2 mL of saliva (ref. 1). In theOragene•DNA vial, the donor’s saliva is mixed with 2 mLof Oragene•DNA fluid, which preserves the DNA in saliva
at room temperature (ref. 2). For applications such as
molecular epidemiology, some scientists would prefer toobtain even greater amounts of DNA. One way to increase
the yield might be to collect an increased volume of saliva.
The purpose of this study was to determine if the DNA
yield from 4 mL of saliva was greater than from 2 mL, andwhether the DNA stability was affected by changing the ratioof Oragene•DNA fluid to sample volume.
Materials and Methods
Saliva collection
Ten donors were enrolled in this study. Each donor wassupplied with two Oragene•DNA vials—one standard vialwith a fill-indicator at the 2 mL level, and a second vial witha modified fill-indicator at the 4 mL level. Donors wereinstructed to alternate spitting between the two vials, until
they were filled to the 2 and 4 mL levels, respectively.

Results
The DNA yields and actual saliva volumes for the 2 and 4
mL collections are shown in Table 1. The DNA yields forthe 2 and 4 mL samples were extrapolated from the amountof DNA in the purified 100-μL aliquots. The average DNAyield was 46.9 and 83.3 μg for the 2 mL and 4 mL collections,respectively. An average of 2.4 mL of saliva was collected inthe vials with the 2 mL fill-indicator, compared to 4.5 mL ofsaliva in the vials with the 4 mL fill-indicator.

DNA yieldfor 4mL
collection(µg)

Actualvolumeof saliva
for 2 mLcollection(mL)

Actualvolumeof saliva
for 4 mL
collection(mL)

16.0

13.6

2.4

4.6

2

45.5

54.4

2.4

5.2

3

25.2

37.4

2.6

3.7

462.9

171.12.3

3.9

571.3

114.2

3.3

5.0

6

51.9

81.4

2.2

4.2

7

10.2

11.4

1.9

4.3

8

33.6

68.8

2.4

4.2

9110.6

188.3

2.2

4.1

10

51.7

92.9

2.7

5.3

Average

46.9

83.3

2.4

4.5

Table 1. DNA yields and actual saliva volumes from thecollection of 2 and 4 mL of saliva.

DNA purification and analysis
From each vial, a 100-μL aliquot was removed and purified
according to the standard Oragene•DNA Purification
Protocol (ref. 3). The DNA yield was determined using
the Fluorescence/DNase (F/D) Assay (ref. 4), a fluorescentquantification assay using SYBR® Green (Molecular Probes).
Agarose gel electrophoresis was used to assess the molecularweight of DNA purified from the Oragene•DNA/saliva
samples stored at room temperature for 98 days. An equalamount of DNA (150 ng) was applied to each well.

DNA yieldfor 2mL
collection(µg)

MDonor 1

Donor 2

Donor 3222

444

M
23.1 kb

Figure 1. Agarose gel of DNA from Donors #1, 2, and 3. The
DNA was purified from the 2 and 4 mL saliva samples after
storage at room temperature for 98 days. A Lambda-Hind III
digest was used as the marker.

Discussion and Conclusions
The DNA yield was increased proportionally to the saliva
volume, with the 4-mL saliva samples yielding an averageof 175% more DNA than the 2-mL samples. In a previous
study with 208 donors (ref. 1), the median DNA yield was
110 ug from 2 mL of saliva. This is higher than the yieldin this study and likely reflects the variability in DNA yieldamong different donors. In terms of DNA preservation, there
was no observable difference in molecular weight between
DNA purified from the 2 or 4 mL saliva samples after
storage at room temperature for more than three months.
In conclusion, the Oragene•DNA Self-Collection Kit can beused to collect and preserve DNA from 4 mL of saliva, witha proportionately higher yield than 2 mL of saliva.
References
1. Birnboim, H.C. (2004). DNA Yield with Oragene™•DNA DNA Genotek. PD-WP001, April 05.
2. Birnboim, H.C. (2004). DNA Stability with Oragene™. DNA Genotek. PD-WP-005,
Dec 04.
3. Laboratory Protocol for Manual Purification of DNA from 0.5mL of Oragene•DNA/Saliva PD-PR-006
4. DNA Quantification Using the Fluorescence/DNase (F/D) Assay. (2004). DNAGenotek.

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