Farklı genomik dna izolasyon yöntemlerinin satureja (labiatae) türlerinde uygulanması

Bitkilerden genomik DNA izolasyonu için farklı protokoller bulunmaktadır. Ancak farklı bitkilerin kimyasal içerikleri de farklı olduğundan birbirine yakın türler için bile özel genomik DNA izolasyon yöntemi gerekli olabilmektedir. Bu çalışmada tıbbi ve aromatik bitkilerden, uçucu yağ ve fenolik bileşiklerce zengin Satureja (Labiatae) cinsi kullanılmıştır. Bu çalışmada üç farklı DNA izolasyon yöntemi uygulanmıştır. Bu yöntemler karşılaştırıldığında bazı değişiklikler yapılarak oluşturulan yöntemin en uygun yöntem olduğuna karar verilmiştir. Bu yöntemle elde edilen gDNA’ da, proteinler, RNA, polisakkaritler, uçucu yağlar, fenoller ve diğer kirleticiler minimal düzeydedir. Bu elde edilen DNA’ların saflığı ve miktarı jel elektroforezi ve UV (A260 /A280 ve A260 / A230) spektroskopik ölçümlerle belirlenmiştir. Ayrıca Rasgele Arttırılmış Polimorfik DNA (RAPD) tekniğiyle DNA’nın PZR tekniklerine uygunluğu gösterilmiştir

There are several methods available for isolation of genomic DNA for plants. However, since different plants have different chemical composition, even close species need specific genomic DNA isolation protocol. In this study, Satureja (Labiatae) genus that is very rich in essential oils and phenolic compounds and one of the aromatic plants has been used. Two different DNA isolation procedures have been tried. A modified procedure was developed after a comparison of two different procedures. Main contaminants for genomic DNA, RNA, polysaccharides, essential oils, phenolic compounds were at minimal level in the genomic DNA. The concentration and purity of the obtained DNA was determined by agarose gel electrophoresis and UV spectroscopy (A260/A280 and A260/230). In addition, the suitability of genomic DNA obtained from different procedure were also checked by RAPD-PCR reactions

Keywords:

DNA extraction Satureja, RAPD-PCR,

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1. P.H.Davis, Flora of Turkey and the East Aegean Islands, (1982), Vol.7,p 314, Üniversity Press, Edinburgh.

2. Başer K.H.C., “Essantial oils from arn,natik plants which are used as herbal teaın Turkey”, Flavours, Fragrances and Essantial Oils.Proceeding of the 13 thInternational Congress of Flavours, Fragrances and Essantial Oils, İstanbul, Turkey(1995)

3. Tümen, G., Kırımer, N., Başer, K.H.C., The essential oils of Satureja L.occurring in Turkey, Proceeding of the 27 th.international symposium on essantial oil, (Franz, C.H.,Mathe, A. And Buuchbauer,G.,EDS.) Allured Publishing Corporation, ViennaAustria, pp.250-254(1997).

4. Başer, K.H.C., Tümen, G., Satıl, F., Kırımer, N. “Comparative Morphological and Chemical Studies on Satureja Species From West Anatolia” Second Balkan Botanical Congress (SBBC), p. 129-132, Ed. By Neriman Özhatay, İstanbul – Turkey. 14-18 May (2000)

5. Lamaison, J.L., Petitjean-Freytet, C., Duband, F., Carnat, A.P.,”Rosmarinic Acid Content and Antioxidant Activity in French”, Fitoterapia, 62, 2:166-171(1991) farmasotik aktivite)

6. Müller-Riebau F., Beger B. And Yegen o., “Chemical composition and fungitoxic properties to phytopathogenic fungi of essantial oils selected aromatic plants growing wild in Turkey”, J. Agric. Food Chem. 43, 2262-2266(1995)(antimkrobial)

7. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski & S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful genetic markers Nucl. Acids. Res. 18:6531-6535

8. Welsh J.,Mc Clelland M (1990) Fingerprinting genomes using PCR with arbitrary primers.Nucl. Acid Res 19:303-306.

9. Southern EM, “Detectionof specific sequences among DNA fragments separated by gel electrophoresis”. J.Mol.Biol., 144:395-404(1975).

10. Aljanabi, S.M., Forget, L., Dookun, A., “An Improved and Rapid Protocol for the Isolation of Polysaccharide and Polyphenol Free Sugarcane DNA” Plant Mol. Bio.Repor. 17:1-8(1999).

11. Porebski S., Bailey L.G. and Baum B.R.,” Modification of a CTAB DNA extraction protokol for plants containing high polysaccharide and polyphenol components” Plant Mol. Biol. Reptr. 15:8-15(1997).

12. Doyle J.J.and Doyle J.L. “A rapid DNA isolation procedure from small quantities of fresh leaf tissue” Phytochem Bull19:11-15(1987)

13. İncirli, A., Bilgiç, H., Akkaya, M. S., "Assessment of Polymorphic AFLP Markers in Triticum durum and Aegilop sp. ", Turk J. Biol. 25, 291-299 (2001).

14. Dellaporta, S. L.,Wood, J., Hicks, J.B., “A Plant DNA Mini Preparation Version II”, Plant Molecular Biology Reporter, Voll 1, (1983) ,.pp:19.

15. Pich, U. And Schubert, I., Minipep Method For İsolation Of DNA From Plants With A High Contentof Polyfenols ",Nüc. Acid. Res.,21, 3328 (1993).

16. Başer K.H.C., Özek T.,Kırımer N. And Tümen G.,“A Comparative Study of The Essantial Oils of Wild and Cultivated Satureja hortensis”, J.Essent.Oil Res.(İn press)

17. Weishing K., Nybom H., Wolf K and Meyer W., “DNA Isolation And Purification. İn:DNA Fingerprinting İn Plants And Fungi, pp 44-59 CRC Press, Boca Raton, Florida (1995).

18. Do N. and Adams R.P. “A Simple Technique For Removing Plant Polysaccharide Contaminants From DNA” BioTechniques 10: 163–166(1991).

19. Sambrook, J. ,Russell, D.W., Maniatis, T, Molecular Cloning A Laboratary Manual. 2ndedition Cold Spring Harbor Laboratory Press Cold Spring Harbor, N. Y. (1989).

20. Öz Aydın S. Bazı Satureja Türlerinin Morfolojik, Moleküler ve Sistematik Yönden Değerlendirilmesi, Doktora Tezi, Balıkesir Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji Eğitimi Anabilim Dalı, Balıkesir, (2004).