Sains Malaysiana 46(6)(2017): 909–915

http://dx.doi.org/10.17576/jsm-2017-4606-10

Kesan Penyimpanan Sampel Air Liur Terhadap Kualiti DNA Genom

(Storage Effect of Saliva Sample on Quality of Genomic DNA)

ROHAYA MEGAT ABDUL WAHAB¹, FARAH AMIRAH MOHD NASRI², INTAN ZARINA ZAINOL ABIDIN³, ZAIDAH ZAINAL ARIFFIN^4,5, MUHAMMAD DAIN YAZID⁶ & SHAHRUL HISHAM ZAINAL ARIFFIN^2*

¹Jabatan Ortodontik, Fakulti Pergigian, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

²Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

³Pusat Sumber Penyelidikan, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Selangor Darul Ehsan, Malaysia.

⁴Jabatan Biologi, Fakulti Sains Gunaan, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia

⁵Atta-ur Rahman, Institut Kajian Ubat Semulajadi, Fakulti Farmasi, Universiti Teknologi MARA, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

⁶Pusat Kejuruteraan Tisu, Tingkat 12, Blok Klinikal, Pusat Perubatan Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, 56000 Cheras, Kuala Lumpur, Wilayah Persekutuan, Malaysia

Diserahkan: 3 Julai 2016/Diterima: 20 Disember 2016

ABSTRAK

Air liur berpotensi menjadi punca DNA yang mudah diambil bagi kajian klinikal kerana tidak invasif berbanding sampel darah. Kajian ini dijalankan untuk memencilkan dan menulenkan DNA genom daripada sampel air liur manusia serta mengkaji kesan penyimpanan terhadap kualiti DNA genom. Sampel air liur (n=5) disimpan dalam penimbal Tris-NaCl EDTA (TNE) pada suhu bilik (25°C) mengikut tempoh masa yang ditetapkan iaitu, segar (tanpa penyimpanan), 1,2,3 dan 4 bulan. Pemencilan dan penulenan DNA dilakukan menggunakan kaedah fenol-kloroform. Seterusnya, PCR telah dijalankan untuk mengetahui ketulenan DNA yang diekstrak menggunakan amplifikasi pada kawasan jujukan beta-globin dan mengenal pasti kehadiran bakteria melalui jujukan yang mengekod 16S rDNA. Keputusan menunjukkan fragmen DNA gen beta-globin manusia hanya berjaya diamplifikasi daripada sampel segar. Sampel air liur yang disimpan dalam penimbal TNE pada suhu bilik tidak mampu menstabilkan DNA genom manusia untuk jangka masa lama dan hanya berkesan untuk tempoh yang singkat iaitu, kurang daripada 1 bulan. Kesimpulannya, hanya sampel air liur segar sahaja yang berupaya memencil DNA genom

Kata kunci: Ketulenan DNA; PCR; penimbal TNE; suhu bilik

ABSTRACT

Saliva is a potential source of DNA easily obtained for clinical studies because it is non-invasive compared to blood samples. This study was carried out to isolate and purify genomic DNA from human saliva sample and to study storage effect on the quality of genomic DNA. Saliva samples (n=5) were kept in Tris-NaCl EDTA (TNE) buffer at room temperature (25°C) according to fixed time period which are; fresh (without storage), 1, 2, 3 and 4 months. Isolation and purification of DNA was carried out using phenol-chloroform method. Next, PCR was conducted to determine the purity of extracted DNA by using amplification of beta-globin sequence region and identify bacterial existence using the sequence that codes for 16S rDNA. Only human beta-globin genomic DNA fragment was successfully amplified from fresh sample. Saliva sample kept in TNE buffer at room temperature was not able to stabilize human genomic DNA at long term and worked for short term storage which was less than a month. In conclusion, fresh saliva sample is needed to isolate genomic DNA.

Keywords: DNA purity; PCR; room temperature; TNE buffer

RUJUKAN

Cheng, T.H., Chen, S.P., Lu, T.C., Chen, W.C., Sher, J.S. & Shieh, Y.S. 2010. Optimal DNA extraction from buccal swab samples. Journal of Medical Sciences 30(4): 149-154.

Chiappin, S., Antonelli, G., Gatti, R. & De Palo, E.F. 2007. Saliva specimen: A new laboratory tool for diagnostic and basic investigation. Clinica Chimica Acta383(1): 30-40.

Cuhadar, S., Koseoglu, M., Atay, A. & Dirican, A. 2013. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochemia Medica 23(1): 70-77.

Gu, F., Li, Y., Zhou, C., Wong, D.T.W., Ho, C.M., Qi, F. & Shi, W. 2009. Bacterial 16S rRNA/rDNA profiling in the liquid phase of human saliva. The Open Dentistry Journal 3: 80-84.

Hebels, D.G.A.J., Georgiadis, P., Keun, H.C., Athersuch, T.J., Vineis, P., Vermeulen, R., Portengen, L. & Ingvar, A. 2013. Performance in omics analyses of blood samples in long-term storage: Opportunities for the exploitation of existing biobanks in environmental health research. Environmental Health Perspectives 121(4): 480-487.

Javaid, M.A., Ahmed, A.S., Durand, R. & Tran, S.D. 2015. Saliva as a diagnostic tool for oral and systemic diseases. Journal of Oral Biology and Craniofacial Research 6: 67-76.

Lima, D.P., Diniz, D.G., Moimaz, S.A., Sumida, D.H. & Okamoto, A.C. 2010. Saliva: Reflection of the body. International Journal of Infectious Diseases 14(3): 184-188.

Luke, R., Khan, S.N., Iqbal, P.S., Soman, R.R., Chakkarayan, J. & Krishnan, V. 2015. Estimation of specific salivary enzymatic biomarkers in individuals with gingivitis and chronic periodontitis: A clinical and biochemical study. Journal of International Oral Health 7(9): 54-57.

Madalli, V.B., Basavaraddi, S.M., Burde, K. & Horatti, P. 2013. Saliva-a diagnostic tool. IOSR Journal of Dental and Medical Sciences 11(6): 96-99.

Malathi, N., Mythili, S. & Vasanthi, H.R. 2014. Salivary diagnostics: A brief review. ISRN dentistry 2014: 1-8.

Maron, J.L. & Johnson, K.L. 2015. Comparative performance analyses of commercially available products for salivary collection and nucleic acid processing in the newborn. Biotechnic & Histochemistry90(8): 581-586.

Mohd Faiz, E., Shahrul Hisham, Z.A., Saiful Anuar, K., Maryati, A.R., Sahidan, S. & Rohaya, M.A.W. 2012. Proteomic analysis of saliva identifies potential biomarkers for orthodontic tooth movement. The Scientific World Journal 2012: Article ID. 647240.

Pham, T.V., Huynh, L.T.T. & Nguyen, T.H. 2015. Modified DNA extraction method for the detection of Aspergillus flavusand Aspergillus parasiticus in dried food. Proceedings of the 5th International Conference on Biomedical Engineering in Vietnam 46: 339-342.

Rathnayake, N., Åkerman, S., Klinge, B., Lundegren, N., Jansson, H., Tryselius, Y., Sorsa, T. & Gustafsson, A. 2013. Salivary biomarkers for detection of systemic diseases. PLoS ONE 8(4): 1-5.

Romanazzi, V., Traversi, D., Lorenzi, E. & Gilli, G. 2015. Effects of freezing storage on the DNA extraction and microbial evaluation from anaerobic digested sludges. BMC Research Notes 8: 1-8.

Rus Dina, R.D., Shahrul Hisham, Z.A., Sahidan, S., Rohaya, M.A.W. & Intan Zarina, Z.A. 2014. Molecular mitochondrial DNA and radiographic approaches for human archaeology identification. Sains Malaysiana43(10): 1523-1535.

Rusterholz, H.P., Ursenbacher, S., Coray, A., Weibel, U. & Baur, B. 2015. DNA quantity and quality in remnants of traffic-killed specimens of an endangered longhorn beetle: A comparison of different methods. Journal of Insect Science 15(1): 1-5.

Shahrul Hisham, Z.A., Mohd Faiz, E., Rohaya, M.A.W., Yosni, B. & Sahidan, S. 2010. Profil aktiviti laktat dehidrogenase, asid fosfatase rintang tartarat dan alkalin fosfatase pada air liur semasa rawatan ortodontik. Sains Malaysiana39(3): 405-412.

Smith, S. & Morin, P.A. 2005. Optimal storage conditions for highly dilute DNA samples: A role for trehalose as a preserving agent. Journal Forensic Science 50(5): 1101-1108.

Spielmann, N. & Wong, D.T. 2011. Saliva: Diagnostics and therapeutic perspectives. Oral Diseases 17(4): 345-354.

Steven, A.W., Barton, E.S. & John, R.M. 2007. Laboratory Investigations in Molecular Biology. Sudbury: Jones and Bartlett Publishers.

Streckfus, C., Bigler, L., Dellinger, T., Dai, X., Kingman, A. & Thigpen, J.T. 2000. The presence of soluble c-erbB-2 in saliva and serum among women with breast carcinoma: A preliminary study. Clinical Cancer Research 6(6): 2363- 2370.

Tina, P., Justin, C.W., Peter, B., Karam, K. & Chamindie, P. 2011. Diagnostic potential of saliva: Current state and future application. Clinical Chemistry 57(5): 675-687.

Wong, D.T. 2006. Salivary diagnostics powered by nanotechnologies, proteomics and genomics. The Journal of the American Dental Association 137(3): 313-321.

Zulham, Y., Ikmal, M.J., Rohaya, M.A.W., Intan Zarina, Z.A., Sahidan, S. & Shahrul Hisham, Z.A. 2013. Orthodontic treatment effects on inflammatory marker profiles in saliva before and after 2 archwire changes. AIP Conference Proceedings 1571: 208-214.

*Pengarang untuk surat-menyurat; email: shahroy8@gmail.com