Sex determination of DNA from Oragene®/saliva samples† using
ReaX™ Mastermix PCR beads
R. Groves, D. Palmer, R. Calder
Q Chip Ltd. Cardiff, UK
2008-09-28

DNA samples collected using the Oragene® self-collection
kit were used in combination with ReaX™ Mastermix
to successfully determine the sex of each DNA sample
donor by employing a qPCR assay for the SRY gene.

Introduction
The number of studies requiring collection ofgenomic DNA from a large number of individuals isincreasing rapidly. Oragene, a self-collection kit for
the isolation, transport and storage of DNA fromsaliva, is ideal for such studies as it provides an easy,
non-invasive method for the collection of large
amounts of high quality DNA.
For sample collection, the donor is asked to abstain
from eating or drinking half an hour before delivering
2 mL of saliva into the Oragene kit. This minimizes
the presence of food particles in the sample. Upon
closing the lid, the Oragene solution is released, andas it mixes with the saliva the DNA is stabilized atambient temperature.
ReaX bead-based mastermixes are enhanced PCR
formulations, which simplify PCR assays and providerobust, reproducible results. Each bead contains
PCR buffer, Magnesium Chloride, dNTPs and Taq
polymerase at final reaction concentrations.
ReaX Mastermix beads are fully optimised for
end-point PCR and a range of real-time reporter
chemistries. ReaX beads can be customized for any
assay via co-encapsulation of specific probes andprimers alongside standard PCR reagents.
A Taqman qPCR assay was developed to detect thepresence of SRY gene1. SRY, or Sex determining
Region Y, encodes the testis determining factor
which initiates male sex determination. Located on
the short arm of the Y chromosome (Yp11.3), it ispredominately found in males and can be utilised to†

distinguish between male- and female-donated DNA
during qPCR. A primer/probe set specific to this locus
was used to produce an amplicon of 80 base pairs.
Hence in male samples, PCR results in amplificationof the SRY gene with corresponding increase in
6-FAM fluorescence. Samples derived from female
donors, which lack the template sequence, produce noincrease in fluorescence and give a negative result.
This technical bulletin demonstrates that thecombination of DNA from Oragene/saliva samples
and ReaX SRY Mastermix beads enables a simplified,streamlined workflow for the performance ofmolecular diagnostic assays. By simplifying DNA
collection from the donor/patient and de-skilling
and accelerating PCR set-up, reliable qPCR results
can be obtained quickly without the need for doctors,
phlebotomists or molecular biology specialists.

Materials and methods
Primers and a 6-FAM hydrolysis probe were designed
from a published description of the SRY gene to produce
an 80 bp target sequence in the qPCR assay. The primer
and probe sequences are given in Figure 1 below.
Forward Primer

5’ GCG ACC CAT GAA CGC ATT 3’

Reverse Primer

5’ AGT TTC GCA TTC TGG AGT
TCT CT 3’

Probe

6-FAM – TGG TCT CGC GAT CAG
AGG CGC – BHQ - 1

Figure 1: SRY primer and probe sequences.

ReaX SRY custom beads were manufactured containing
the following PCR components within each bead.
••

10 pM of each primer
0.1 pM of probe

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

(outside the USA)(in the USA)

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••••

800 µM dNTPs (200 µM of each)
5 mM MgCl2
1x ReaX PCR buffer
1 unit of HotStart Taq polymerase

Saliva samples were obtained from 100 donors.
Collection and purification of DNA was carried out
according to the prepIT•L2P protocol2 for use with
Oragene/saliva sample. For each of 100 samples, a25 µL PCR reaction was set up, containing purified
DNA (0.5 µL diluted with 24 µL of ddH2O). Each DNA
dilution was then added to a single pre-welled ReaX
bead and each tube was sealed with a tube cap andthen placed in the Mastercycler® Realplex2 qPCR
thermal cycle (www.eppendorf.com).

Inconclusive

Thermocycle program:

Positive

94°C – 300 secs

Negative

95°C – 15 secs
58°C – 60 secs}X39

Figure 3: ReaX beads qPCR results represented diagrammatically toeasily identify positive/negative results and therefore the sex of eachsample of DNA from Oragene/saliva samples.

Results
The presence or absence of an amplification curvefor each Oragene/saliva sample was recorded.
Results are shown in Figures 2 and 3. Note thatthe concentration of DNA in each sample was notstandardized prior to PCR.

Conclusions
The results obtained were 100% concordant for all of
the Oragene/saliva samples tested with the SRY ReaX
bead qPCR assay.

Figure 2: DNA from Oragene/saliva samples - ReaX amplification curves.
Red – amplification of the SRY gene occurs indicating that the DNA
sample is derived from a male individual.
Green – no amplification curve is produced therefore no SRY gene ispresent in the DNA sample indicating that the DNA sample is derived
from a female individual.
References
12

Clepet C., Schafer A.J., Sinclair A.H., Palmer M.S., Lovell-Badge R., and Goodfellow P.N. (1993). The human SRY transcript.
Hum Mol Genet 2:2007‑2012.
Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.

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.

MK-AN-018 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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