Pulsed-Field Jel Elektroforez (PFGE) Metodu ve Akuatik Organizmalarda Kullanımı

Öz

PFGE (Pulsed-Field Jel Electroforez), akuakültürde salgın hastalıklara yol açan izolatların ilişkilendirilmelerinde anahtar rol oynayan bir metottur. Teknik, 10 megabaz çifti (Mbç) kadar geniş molekül ağırlığına sahip DNA parçalarının etkin bir biçimde ayrışmasına olanak sağlamıştır. PFGE, çeşitli patojenlerin epidemiyolojisi, genom büyüklüklerinin belirlenmesi, bakteri kromozomlarının fiziksel ve genetik haritalandırılmalarının gerçekleştirilmesi gibi konularda son yıllarda sıkça kullanılan ve altın standart olarak kabul gören bir metottur. Agaroz içerisindeki bakteri hücresinden elde edilen genomik DNA’nın, uygun enzim ile kesimi sonucu oluşan bant desenlerinin yorumlanması şeklinde gerçekleştirilir. Bu derlemede, PFGE’nin genel özellikleri, çalışma prensibi, tipleri, genel ve akuakültürdeki kullanımından söz edilmiştir.

Anahtar Kelimeler

• Akuakültür, Epidemiyoloji, PFGE, Kesici enzimler

Method of Pulsed-Field Gel Electroforesis (PFGE) and Its Usage in Aquatic organisms

Abstract

PFGE (Pulsed-Field Gel Electroforesis) played a key role in determining the outbreak-associated isolates in aquaculture. The technique has enable to separate large DNA fragments of up to 10 megabase pairs (Mb) effectively. In recent years PFGE, is a method that comonly used and accepted as the gold standard for epidemiology of various pathogen, determining to genome size and constructing the physical and genetic map of the chromosome of bacteria. PFGE is performed: Genomic DNA is derived from bacterial cell in agarose, cut with the appropriate enzyme and band paterns are described. In this review, the general characteristics of PFGE, prenciples of experiment, types of PFGE, the use of PFGE in general and aquaculture are introduced.

Keywords

• Aquaculture, Epidemiology, PFGE, Restriction enzymes

References

1. Basım, E., Basım, H., 2001. Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology. Turk. J. Biol 25: 405-418.
2. Birren, B.W., Lai, E., Clark, S.M., Houd, L., Simon, M.I., 1988. Optimized conditions for pulsedfield gel electrophoretic separations of DNA. Nucl. Acids Res., 16: 7563-7581.
3. Burmeister, M., 1992. Methods in Molecular Biology. Eds. M. Burmeister, L. Ulanovsky, New Jersey, Humana Press, 259-284.
4. Carle, G.F., Olson, M.V., 1984. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucl. Acids Res., 14: 5647-5663.
5. Carle, G.P., Frank, M., Olsen, M.V., 1986. Electrophoretic separations of large DNA molecules by periodic inversion of the electnc field. Science, 232: 65-68.
6. Chu, G., Vollrath, D., Davis, R.W., 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science, 234:1582-1585.
7. Correia, A., Martin, J.F., Castro, J.M., 1994. Pulsed-field gel electrophoresis analysis of the genome of aminoacid producing Corynebacteria: Chromosome sizes and diversity of restriction patterns. Microbiology, 140: 2841- 2847.
8. Gardiner, K. 1991. Pulsed-Field Gel Electrophoresis. Analytical Chemistry, 63: 658-665.

9. Gardiner, K., Patterson, D., 1988. Transverse alternating electrophoresis. Nature, 331:371-372.
10. Kawanishi, A., Kojima, A., Ishihara, K., Esaki, H., Kijima, M., Takahashi, T., 2005. Drug Resistance and Pulsed-Field Gel Electrophoresis Patterns of Lactococcus garvieae Isolates from Cultured Seriola (Yellowtail, Amberjack and Kingfish) in Japan. Lett. Appl. Microbiol., 40: 322-328.
11. Lai, E., Birren, B.W., Clark, S.M., Simon, M.I., Hood, L., 1989. Pulsed-Field Gel Electrophoresis. Biotechniques, 7: 34-42.
12. Levene, S.D., 1992. Methods in Molecular Biology, Pulsed-Field Gel Electrophoresis. Eds. M. Burmeister and L. Ulanovsky, Totowa, NJ, The Humana Press, 345-365.
13. Mc Ellistrem, M.E. Stout, J., H. Harison, L., 2000. Simplifield Protocol for Pulsed-Field Gel Electrophoresis Analysis of Streptococcus pneumoniae. Journal of Clinical Microbiology. 351-353.
14. Murray, P.R., Rosenthal, K.S., Kobayashi, G.S., Pfaller, M.A., 1998. Medical Microbiology. Mosby Inc., St. Louis, 140-144.
15. Olive, B.M., Bean, P., 1999. Principles and Aplication of Methods DNA Based Typing of Microorganism. J. Clin. Microbiol., 37; 1661-1669.
16. Schwartz, D.C., Saffran, W., Welsh, J., Haas, R., Goldenberg, M., Cantor, C.R., 1982. New techniques for purifying large DNAs and studying their prioreties and packaging. Cold Spring Harbor Symposia on Quantitative Biology, XLVII: 189-195.
17. Schwartz, D.C., Cantor, C.R., 1984. Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis. Cell, 37:67–75.
18. Smith, C.L., Klco, S.R., Cantor, C.R., 1988. Genome analysis a practical approach. Edited by K. E. Davis, Washington D.C., IRL Press, Oxford, 41-72.
19. Steward, G., Furst, A., Avdalovic, N., 1988. Transferse Alternating Field Electrophoresis (TAFE). Biotechniques, 6: 68-73.
20. Swaminathan, B., Mathar, G.M., 1993. Moleculer Typng Methods. In Persing, D.H., Smith T.F., Tenover F.C., White, T.C. (eds.). Diagnostic Molecular Microbiology: Prenciple and Aplication. ASM Pres, 26-50.
21. Tejedor, J.L., Vela, A.I., Gibello, A., Casamayor, A., Dominguez, L., Fernandez, J.F., 2011. A Genetic Comparison of Pig, Cow and Trout Isolates of Lactococcus garvieae by PFGE Analysis. Letters in Applied Microbiology, 53: 614–619.
22. Tenover, F.C., Albeit, R.D., Georing R.E., 1995. Interprteing Cromosomal DNA Restriction Pattern Produced by Pulse-Field Gel Electroforesis: Criteria for Bacterial Straing Typing. J. Clin. Microbiol., 33: 2233-2239.
23. Turabelidze, D., Kotetishvili, M., Kreger, A., J. Morris, J.G., Sulakvelidze, A., 2000. Improved Pulse-Field Gel Elektroforesis for Typing Vancomycin-Resistant Enterococci. Journal of Clin. Microbiol., 4242-4245.
24. Türe, M., Işıdan, H., Savaş, H., Kutlu, İ., 2012. PFGE Metodu Kullanılarak Lactococcus garvieae’nin Genetik Çeşitliliğinin ve Yayılımının Belirlenmesi Projesi. TAGEM/HS/10/09/02/179, s:61.
25. URL1.http://www.pulsenetinternational.org/SiteCollectionDocuments/pfge/PFGE_troubleshooting. pdf, Kara Cooper. PFGE: General Overview and Troubleshooting Tips. Erişim tarihi: 25.03.2011.
26. URL2. http://web.inonu.edu.tr/~iozerol/rdurmaz/UygMolMikr/161.pdf. Durmaz, B. ve Durmaz, R. Erişim tarihi: 18.06.2012.
27. Vela, A.I., Vazquez, J., Gibello, A., Blanco, M.M., Moreno, M.A., Liebana, P., 2000. Phenotypic and Genetic Characterization of Lactococcus garvieae Isolated in Spain from Lactococcosis Outbreaks in Comparison with Isolates of Other Countries and Sources. J. Clin. Microbiol., 38: 3791-3795.
28. Ziegler, A., Vols, A., 1992. Methods in Molecular Biology, Pulsed-field gel electrophoresis. Eds. M. Burmeister, L. Ulanovsky, Totawa, NJ, Humana Press, 63-72.