Clinical Oral Investigations

, Volume 17, Issue 7, pp 1719–1725 | Cite as

Evaluation of the long-term storage stability of saliva as a source of human DNA

  • Robert P. Anthonappa
  • Nigel M. King
  • A. Bakr M. Rabie
Original Article

Abstract

Objectives

The objectives of this paper are to determine the storage stability of saliva at 37 °C over an 18-month period, and its influence on the DNA yield, purity, PCR protocols and genotyping efficacy.

Materials and methods

Of the 60 participants, blood samples were obtained from 10 and saliva from 50. Samples were subjected to different storage conditions: DNA extracted immediately; DNA extracted following storage at 37 °C for 1, 6, 12 and 18 months. Subsequently, DNA yield, OD260/280 and OD260/230 ratios were measured. The isolated DNA was used to amplify exons 0–7 of the RUNX2 gene and subsequently sequenced. Furthermore, 25 SNPs were genotyped.

Results

The mean DNA yield, OD260/280 and OD260/230 ratios obtained from blood were 67.4 ng/μl, 1.8 ± 0.05 and 1.8 ± 0.4 respectively. DNA yield obtained from saliva was significantly higher than blood (p < 0.0001), ranging from 97.4 to 125.8 ng/μl while the OD260/280 ratio ranged from 1.8 ± 0.13 to 1.9 ± 0.1. The success rates for the 25 SNPs ranged from 98 to 100 % for blood and 96–99 % for saliva samples with the genotype frequencies in Hardy–Weinberg equilibrium (>0.01).

Conclusions

Saliva can be stored at 37 °C for 18 months without compromising its quality and ability to endure genetic analyses.

Clinical relevance

Saliva is a viable source of human DNA to facilitate the feasibility of large-scale genetic studies.

Keywords

Saliva Blood DNA RUNX2 

References

  1. 1.
    Ng DP, Koh D, Choo S, Chia KS (2006) Saliva as a viable alternative source of human genomic DNA in genetic epidemiology. Clin Chim Acta 367:81–85PubMedCrossRefGoogle Scholar
  2. 2.
    Ng DP, Koh D, Choo S, Ng V, Fu QY (2004) Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva. Clin Chim Acta 343:191–194PubMedCrossRefGoogle Scholar
  3. 3.
    Quinque D, Kittler R, Kayser M, Stoneking M, Nasidze I (2006) Evaluation of saliva as a source of human DNA for population and association studies. Anal Biochem 353:272–277PubMedCrossRefGoogle Scholar
  4. 4.
    Rogers NL, Cole SA, Lan HC, Crossa A, Demerath EW (2007) New saliva DNA collection method compared to buccal cell collection techniques for epidemiological studies. Am J Hum Biol 19:319–326PubMedCrossRefGoogle Scholar
  5. 5.
    Mulot C, Stücker I, Clavel J, Beaune P, Loriot MA (2005) Collection of human genomic DNA from buccal cells for genetics studies: comparison between cytobrush, mouthwash, and treated card. J Biomed Biotechnol 3:291–296CrossRefGoogle Scholar
  6. 6.
    García-Closas M, Egan KM, Abruzzo J, Newcomb PA, Titus-Ernstoff L, Franklin T et al (2001) Collection of genomic DNA from adults in epidemiological studies by buccal cytobrush and mouthwash. Cancer Epidemiol Biomarkers Prev 6:687–696Google Scholar
  7. 7.
    Cozier YC, Palmer JR, Rosenberg L (2004) Comparison of methods for collection of DNA samples by mail in the Black Women’s Health Study. Ann Epidemiol 2:117–122CrossRefGoogle Scholar
  8. 8.
    Luan JA, Wong MY, Day NE, Wareham NJ (2001) Sample size determination for studies of gene–environment interaction. Int J Epidemiol 30:1035–1040PubMedCrossRefGoogle Scholar
  9. 9.
    Holland NT, Smith MT, Eskenazi B, Bastaki M (2003) Biological sample collection and processing for molecular epidemiological studies. Mutat Res 543:217–234PubMedCrossRefGoogle Scholar
  10. 10.
    Xuan D, Li S, Zhang X, Hu F, Lin L, Wang C et al (2008) Mutations in the RUNX2 gene in Chinese patients with cleidocranial dysplasia. Ann Clin Lab Sci 38:15–24PubMedGoogle Scholar
  11. 11.
    Phillips MS, Lawrence R, Sachidanandam R, Morris AP, Balding DJ, Donaldson MA et al (2003) Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots. Nat Genet 33:382–387PubMedCrossRefGoogle Scholar
  12. 12.
    Halsall A, Ravetto P, Reyes Y, Thelwell N, Davidson A, Gaut R et al (2008) The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4 degrees C for up to 24 h. Int J Epidemiol 37(Suppl 1):i7–i10PubMedCrossRefGoogle Scholar
  13. 13.
    Visvikis S, Schlenck A, Maurice M (1998) DNA extraction and stability of epidemiological studies. Clin Chem Lab Med 36:551–555PubMedCrossRefGoogle Scholar
  14. 14.
    Dawes C (2003) Estimates, from salivary analyses, of the turnover time of the oral mucosal epithelium in humans and the number of bacteria in an edentulous mouth. Arch Oral Biol 48:329–336PubMedCrossRefGoogle Scholar
  15. 15.
    Halsall A, Ravetto P, Reyes Y, Thelwell N, Davidson A, Gaut R et al (2008) The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4 degrees C for up to 24 h. Int J Epidemiol 37:7–10CrossRefGoogle Scholar
  16. 16.
    Rylander-Rudqvist T, Håkansson N, Tybring G, Wolk A (2006) Quality and quantity of saliva DNA obtained from the self-administrated oragene method—a pilot study on the cohort of Swedish men. Cancer Epidemiol Biomarkers Prev 15:1742–1745PubMedCrossRefGoogle Scholar
  17. 17.
    Chartier J, Birnboim H (2004) Bacterial DNA content with OrageneTM. DNA Genotek, OttawaGoogle Scholar
  18. 18.
    van Schie RC, Wilson ME (1997) Saliva: a convenient source of DNA for analysis of bi-allelic polymorphisms of Fc gamma receptor IIA (CD32) and Fc gamma receptor IIIB (CD16). J Immunol Methods 208:91–101PubMedCrossRefGoogle Scholar
  19. 19.
    Andrisin TE, Humma LM, Johnson JA (2002) Collection of genomic DNA by the non-invasive mouthwash method for use in pharmacogenetic studies. Pharmacotherapy 22:954–960PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Robert P. Anthonappa
    • 1
    • 3
  • Nigel M. King
    • 1
  • A. Bakr M. Rabie
    • 2
  1. 1.Paediatric Dentistry, School of DentistryThe University of Western AustraliaPerthAustralia
  2. 2.Paediatric Dentistry and Orthodontics, Faculty of DentistryThe University of Hong KongHong KongChina
  3. 3.Paediatric Dentistry, School of DentistryOral Health Centre of Western AustraliaNedlandsAustralia

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