Oragene®/saliva samples† and DNA sequencing with ABI PRISM®
J. Chartier

DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

DNA from Oragene®/saliva samples is reliably
sequenced using the ABI PRISM® 377 DNA sequencer.

Introduction
Blood leukocytes are the traditional source of DNA
for molecular testing, but collection is invasive andspecimens require infectious precautions. Buccalswabs are a less invasive method of collecting DNA
for sequencing applications such as HLA typing andprelingual deafness screening1, 2 but they tend tohave an appreciable failure rate3. Oragene is anon-invasive DNA self-collection kit from saliva thatprovides significantly higher DNA yields than buccal
swabs4. The purpose of this study was to investigate
the suitability of DNA from Oragene/saliva samples
for DNA sequencing.

Materials and methods
DNA collection
Saliva samples were collected from 5 donors using
the Oragene kit. Collection and purification of DNA
was carried out according to the prepIT™•L2P
purification protocol5.

Primer design
PCR primers for the human Thymidylate Synthetase(TYMS) gene were designed based on the publicly
available DNA sequence (GenBank accession no.
AP001178). The primers generate a 560 bp fragment.
Table 1 above shows the primer sequences.

Primer name

Sequence (5’ - 3’)

TS 560-forward

ATGCTTAGTAGGCAATTCTG

TS 560-reverse

TTTGGTTGTCAGCAGAGG

Table 1: PCR primers for the Thymidylate Synthetase gene.

DNA sequencing
Purified DNA from each of the 5 Oragene/saliva
samples was used as the template for PCR with the TS
560 primers. The PCR products were sequenced inboth directions by Cortec DNA Service Laboratories(Kingston, ON) using an ABI PRISM 377 DNA
sequencer (Applied Biosystems) and the DYEnamic™
ET dye terminator kit (Amersham Biosciences). Prior
to sequencing, the PCR products were purified using
the microCLEAN™ DNA clean‑up reagent (Microzone).

Results
DNA sequencing results were aligned using
CLUSTAL W (Version 1.83), a multiple sequencealignment program. All 5 samples correctly aligned
to the original human Thymidylate Synthetase genesequence. Figure 1 shows a representative ABIPRISM sequencing read-out.

Figure 1: ABI PRISM sequencing read-out.
†Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

Some DNA Genotek products may not be available in all geographic regions, contact your sales representative for details.

www.dnagenotek.com • support@dnagenotek.com

Discussion and conclusions
Oragene is a non-invasive and reliable method ofcollecting DNA from saliva. DNA from Oragene/saliva samples works well for DNA sequencing withthe ABI PRISM 377 DNA sequencer.

References
123
45

Liu, C., Chu, C., Lee, Y., Shi, Y. and Lin, M. (2004). Identification of two novel HLA-DRB1 alleles, DRB1*0903 and DRB1*1145.
Tissue Antigens. 64: 99–101.
Sugata, A., et al. (2002). High-throughput screening for GJB2 mutations – its clinical application to genetic testing in prelingual
deafness screening for GJB2 mutations. Auris, Nasus, Larynx. 29: 231-239.
Elit, L., Jack, E., Kwan, E., Baigal, G. and Narod S. (2001). A unique BRCA1 mutation identified in Mongolia. International Journalof Gynecological Cancer. 11: 241-243.
Birnboim, H.C. (2004). DNA yield with an Oragene self-collection kit. DNA Genotek. PD-WP-001.
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-010 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

2