Impact of population and laboratory methods on DNA yieldand variability
Total DNA yield from Oragene®/saliva samples† canvary due to intra- and inter-donor variability, as wellas the DNA extraction and quantification methods
chosen. A comparative analysis of total DNA yield frompeer-reviewed publications and in-house experiments
is presented in this white paper to highlight the impactof population and laboratory method selection on
final interpretation of data. Using comparative data,
the average DNA yield from 2 mL of saliva extractedwith prepIT®•L2P is 56 µg when quantified usingfluorescence, and the average total nucleic acid yieldis 129 µg when quantified by UV spectroscopy.

Introduction
In order to reduce costs and maximize donor
compliance, researchers are turning to non-invasive,
easy self-collection methods for obtaining genetic
samples. Traditionally blood has been considered
the standard sample for genetic testing, however theinvasiveness and cost associated with this sample
type has been a barrier for widespread use in various
applications. Oral samples have been proven to
be a reliable and accessible source of DNA for
use in clinical diagnostics, genetic research andpersonalized medicine. On average, the amountof DNA per millilitre of sample is similar between
blood and saliva. The amount and quality of DNA
recovered from oral samples can vary depending on
the collection method used as well as the extraction
and quantification protocols. The Oragene selfcollection kit is a non-invasive, simple to use device
which makes it suitable for collecting samples in thefield, in clinics and at home. This paper summarizes
the total DNA yield from Oragene/saliva samples asreported in peer-reviewed publications, conference
presentations and technical literature and discusses
possible explanations for the variations seen
between studies.

†DNA quantification methodology
The most important factor to consider whencomparing total DNA yield from different studies is
the DNA quantification method used to determine
sample yield. Different methods of quantification
result in dramatic differences in reported yields.
UV spectroscopy is perhaps the most commonlyused method to quantify DNA. While inexpensive
and convenient, it suffers from a lack of specificity
in the nucleic acids it quantifies. UV spectroscopy
cannot distinguish between the DNA and RNAcontent of the purified extracted sample, therefore
the total yield reported will represent the total
nucleic acid content, not just the DNA content.
Comparatively, fluorescence-based methods ofquantification, such as PicoGreen®, quantify only thedouble-stranded DNA resulting in a more accurate
and specific measurement of DNA in a sample.
Several studies have compared UV spectroscopy
to PicoGreen and reported significant differences
between the DNA yields reported by the two methods.
Data from these studies show that DNA quantification
by absorbance are approximately 2-3 fold higherthan those reported by PicoGreen quantification(Table 1: Studies 5,11,17,18 and 21; Table 2). Table 2
demonstrates the average DNA yield, regardless ofpurification method, is overestimated by 2 foldwhen relying on UV spectroscopy for quantification.
The data underscores the importance of complete RNA
removal from a sample if UV spectroscopy is to be
used for accurate DNA quantification. However, since
many downstream applications use only doublestranded DNA as a starting source, removal of RNA
is an unnecessary additional step that can actually
introduce variability and result in a loss of DNA.
A simpler and more practical approach to accurately
quantifying DNA yield, as opposed to total nucleic
acid content, is quantification by fluorescence.

Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER(outside the USA)(in the USA)

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DNA extraction methodology
The protocol methodology used to extract the DNA
from a raw Oragene/saliva sample has a direct impact
on the DNA yield. The most common methodsof DNA extraction include ethanol precipitation,
spin columns and magnetic bead-based systems.
Each method has advantages and disadvantages
with respect to simplicity, quality and quantityof DNA obtained.
Magnetic bead-based and spin column-based
methods are subject to the same limitations: binding
capacity and binding efficiency. Binding capacity is
the maximum amount of DNA that can be absorbed
and is dependent upon the physical properties of thebeads or filters used. Binding efficiency describes how
well DNA will bind to the beads/filters and is affectedby factors such as pH, salt concentration and thepresence of competing macromolecules. DNA in asample that is in excess of the binding capacity of thematrix being used, or that is unable to bind due topoor binding efficiency, will be lost during purification.
Oragene/saliva samples are compatible with thesepurification methods (Studies 16-29). The average
DNA yield, as measured using fluorescence, using
magnetic bead-based or spin column-based methods
is 31.2 µg (Table 2). This represents a 1.8 folddecrease in comparison to samples purified using
the ethanol-based prepIT•L2P.
Ethanol precipitation-based extraction methods(i.e., prepIT•L2P or Puregene®) are not subject tobinding capacities and efficiencies; virtually all
high molecular weight DNA in the sample will be
precipitated and collected by centrifugation. As aresult, precipitation-based methods tend to provide
higher recoveries than column- or bead-based systems.
In Table 2 the summary of study results shows theaverage yield of total nucleic acids, by fluorescence(UV absorbance), from precipitation-based extractions(Studies 1-18, 21-24) was 56.3 µg (128.7 µg) purifiedusing prepIT•L2P and 40.3 µg (101.2 µg) purified using
Autopure LS®/Puregene. This compared to an average
yield of 31.2 µg (58.4 µg) using bead/column methods(Studies 16-27). PrepIT•L2P is optimized for
purification of Oragene/saliva samples and is not
subject to loss of DNA pellets during automation.
In spite of the differences in yield and cost whencompared to ethanol precipitation, column- andbead-based extraction methods may provide
sufficient yield for downstream analyses and have

PD-WP-00031 Issue 1/2013-01© 2013 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

the added advantages of being simpler and quicker
protocols. However, the impact on yield must be
taken into consideration when comparing studies
using oral samples and when determining theexperimental design.

Intra-donor variability
The DNA content of the saliva of a particular
individual may vary naturally over the course of a day
and may be influenced by particular eating or drinking
habits. For this reason, the instructions for collecting
an Oragene/saliva sample using an self-collection kit
include the precaution to wait 30 minutes after eating,drinking, smoking or chewing gum before giving asaliva sample.
Two internal studies (DNA Genotek, 2008,unpublished data) investigated the intra-donor
variability of saliva samples. The first study (Table 1,Study 14) included 33 donors and 3 samples/donor.
The average yield per donor was 62.3 µg, however
variability within donors had an average standard
deviation of ±21.7 (range ±1-75 SD). The second
study (Table 1, Study 15) investigated the variation
among 41 donors with 6 samples/donor for a total of
246 samples. Here the average yield was 49.9 µg withan average ±25.3 SD (range ±3-95 SD). The data
highlights some considerations that need to be taken
into account when comparing yield data between
different collections.

Variation within a population
Total DNA yield from saliva varies between individuals
making the study population an importantconsideration when assessing average yield of an
entire study. The DNA yield from a population ofhealthy adults can vary from a few micrograms toalmost 400 µg in 2 mL of saliva, with 95% of donors
falling between 10 and 165 µg (Table 1, Study 13).
When collecting from a more restricted population
the average yield and yield range may vary.
For example, in a study of transplant recipients
conducted by Chakkera et al.4, the average DNAyield from 2 mL of saliva was 13.3 µg compared toaverage yields of >50 µg with healthy populations(Studies 1-15). Population based factors must be taken
into account when evaluating DNA yield from saliva.

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2Table 1: Summary of average DNA yield from Oragene self-collection kits by study
Mean yield for 2 mL saliva
StudynumberNumberof samples

DNA extraction method

UV Spectroscopy

12101

prepIT•L2P

167.6

12291

prepIT•L2P

187.2

2321

prepIT•L2P

95.2

3491

prepIT•L2P

13.3

45539

prepIT•L2P

155.2

672

prepIT•L2P

86.4

7790

prepIT•L2P

817

prepIT•L2P

154.9

Fluorescence

82.2

Reference
number

56NA

78966

prepIT•L2P

152

91010

prepIT•L2PNA101120

prepIT•L2P

139.3012208

prepIT•L2P

13450

1499

15246

168172463.65

11110*,**

12prepIT•L2P

58.5

13prepIT•L2P

62.28

14prepIT•L2P

49.87

15

prepIT•L2P224

Qiagen QIAamp82

prepIT•L2P

40.4

32.8

Promega74

70.9

prepIT•L2P
1860

16

1744.5

GeneFind

75.5

64.7

DNAdvance

55.4

33.0

18

1920

prepIT•C2D

21.0

15.8

1920

20

prepIT•C2D

17.7

13.85

2021

NA

Gentra Puregene

NA21

22

16

Gentra Puregene

85.5

22

2390

Autopure LS (Puregene)

135.9

24

16

Autopure LS (Puregene)

82.3

25

10

AutoGenFlex

122.4

25

2696

Promega Magazorb DNA Mini (Tecan Freedom EVO)67.2262718Qiagen BioRobot EZ1 Tissue Kit

2820Magtration 12GC

76.8282916

Promega Maxwell 16 Tissue Kit

56 (NA)

2940.3232464.0*,**27

NA = information not published by author* Median, not mean** F/D Assay

PD-WP-00031 Issue 1/2013-01© 2013 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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Table 2: Overall DNA yield from Oragene self-collection kits
Mean yield for 2 mL saliva (µg)

DNA extraction
methodUV spectroscopy

Fluorescence

Overestimation byUV spectroscopy (µg)

Fold overestimation byUV spectroscopy

prepIT•L2P

128.7

56.3

72.4

2.3

Autopure

101.2

40.3

60.9

2.5

Beads/Columns

58.4

31.2

27.2

1.9

All methods

96.1

42.6

53.5

2.3

Conclusion
When evaluating results from an Oragene/saliva
collection, or when trying to compare data frommultiple collections or studies, it is important totake the following parameters into consideration:
the population being sampled (age, health status,demographics), the time of collection, adherence

to collection instructions, purification andquantification methods. Highest yields are observed
when purifying with prepIT•L2P. Quantification using
fluorescence provides an accurate and DNA specificquantification while quantification by UV
spectroscopy tends to overestimate the amountof DNA by 2-3 fold.

References
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14151617181920
21
2223
242526272829

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Viltrop T et al. Comparison of DNA extraction methods for multiplex polymerase chain reaction. Anal Biochem. 398(2):260-262 (2010).
Chakkera HA et al. Pilot study: association of traditional and genetic risk factors and new-onset diabetes mellitus following kidney transplantation. Transplant Proc.
41(10):4172‑4177 (2009).
Nishita DM et al. Clinical trial participant characteristics and saliva and DNA metrics. BMC Med Res Methodol. 9:71 (2009).
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Cancer Epidemiol Biomarkers Prev. 16(10):2072-2076 (2007).
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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 and prepIT are registered trademarks 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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