SNP genotyping of DNA from Oragene®/saliva samples† with SNPstream®
M. Van Oene‡, S. Alic‡, A. Jackson**, and P. Lem**‡ Ellipsis Biotherapeutics, Toronto, Ontario, Canada** DNA Genotek., Ottawa, Ontario, Canada
2006-06-29

Genomic DNA collected from saliva with the Oragene®
self-collection kit is successfully genotyped with theSNPstream® genotyping system, and gives the same
results as DNA isolated from whole blood.

Introduction
The GenomeLab™ SNPstream genotyping system(Beckman Coulter) combines the features of solidphase chip array, primer extension assay, anduniversal tags for the genotyping of single-nucleotide
polymorphisms (SNPs). The instrument allows thegenotyping of up to 1,000,000 SNP genotypes per day,with 96% sample call rates, and >99% concordancewith reference genotypes1. Sources of genomic DNA
such as whole blood2 and highly degraded forensic
samples3 have been validated with the SNPstreamsystem. The purpose of this study was to compare
the SNP genotyping results between paired bloodand saliva samples collected with the Oragene
self-collection kit.

Materials and methods
DNA collection
Paired saliva and whole blood samples were collectedfrom 25 donors. The saliva samples were collectedusing the Oragene self-collection kit, and purified
according to the prepIT™•L2P purification protocol4.
Genomic DNA was purified from the whole blood
samples using the QIAamp DNA blood mini kit(Qiagen). Purified DNA was quantified by fluorescencewith SYBR® Green I dye (Molecular Probes)5.

†SNP genotyping
The SNPstream genotyping assay was performed
according to methods previously described by Bellet al.1. In brief, 4 ng of each DNA sample was used
for 12-plex PCR amplification. The PCR-amplified
fragments were treated with a cocktail of exonuclease I
and shrimp alkaline phosphatase to degrade
unincorporated PCR primers and dNTPs. Tagged
extension primers were extended using single
TAMRA- or BODIPY-Fluorescein-labeled nucleotide
terminator reactions, and spatially resolved byhybridization to the complementary oligonucleotide
tag on the SNPware® Tag Array (384-well microplateformat). The individual SNPs within the multiplex
were identified according to the position of thearrayed oligonucleotides within each well. Basedon the relative fluorescent intensities for each spot,individual sample genotype data was generated
and computer-processed for graphical review.

Results
There was 100% concordance for the genotyping calls
between the paired blood and saliva samples, withcomparable signal intensity and overall data quality.
Initially, one of the saliva samples had weak signal
intensity, however successful genotyping was achievedon repeat analysis. Figure 1 shows representativegraphical output of the TAMRA/BODIPY fluorescencesignals obtained for SNP #5 of 12.

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

(outside the USA)(in the USA)

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(A) Saliva

Discussion and conclusions
DNA from the Oragene/saliva samples gave clear
genotyping calls with the SNPstream genotyping
system and had 100% concordance with the paired
blood samples. These results are in agreement withother studies showing that genomic DNA from oral
sources gives the same genotyping results as DNA fromwhole blood6, 7. In summary, genomic DNA fromsaliva may be collected in a non-invasive mannerwith the Oragene self-collection kit, and accurately
genotyped with the SNPstream genotyping system.

About Ellipsis Biotherapeutics(B) Whole Blood

Ellipsis Biotherapeutics Corporation provides
custom SNP genotyping services using state-ofthe‑art technologies such as the GenomeLab
SNPstream genotyping system. For more
information, please visit www.ellipsisbio.com,
or contact genotyping@ellipsisbio.com.

Figure 1: SNPstream graphical output of the TAMRA/BODIPY
fluorescence signals obtained for SNP #5 of 12. Each saliva (A) orwhole blood (B) sample is depicted by green, orange or blue open
circles within the associated genotype clusters.

References
1234567Bell, P., et al. (2002). SNPstream® UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery.
BioTechniques. 32: S70-S77.
Denomme, G. and Van Oene, M. (2005). High-throughput multiplex single-nucleotide polymorphism analysis for red cell andplatelet antigen genotypes. Transfusion. 45: 660-666.
Budowle, B. (2004). SNP typing strategies. Forensic Science International. 146S: S139–S142.
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 dyeand a micro-plate reader. DNA Genotek. PD-PR-075.
Zheng, S., Ma, X., Buffler, P., Smith, M. and Wiencke, J. (2001). Whole genome amplification increases the efficiency and validityof buccal cell genotyping in pediatric populations. Cancer Epidemiology, Biomarkers & Prevention. 10: 697–700.
de Vries, H., et al. (1996). Validation of the determination of ∆F508 mutations of the cystic fibrosis gene in over 11000 mouthwashes.
Human Genetics. 97: 334–336.

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Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
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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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