Serpil KAHYA, Esra BUYUKCANGAZ, K. Tayfun CARLI

Polimeraz Zincir Reaksiyonu (PCR) Optimizasyonu

Polimeraz zincir reaksiyonu (PCR), bir deoksiribo nükleik asit (DNA) zincirinin bilinen iki parçası arasında uzanan özel bir DNA bölümünün enzimatik olarak çoğaltıldığı in vitro bir teknik olarak her geçen gün yaygınlaşmaktadır. PCR metodu, teşhis, epidemiyolojik ve DNA miktarı belirleme çalışmaları gibi birçok amaçla kullanılmakta ve geliştirilmeye devam edilmektedir. PCR’ın kullanıldığı tüm alanlarda meydana gelen gelişmelerden, mikrobiyoloji alanı da önemli oranda payını alarak gelişmeye devam etmektedir. İnsan ve hayvan kaynaklı patojenik mikroorganizmalar için pek çok amaçla kullanılan PCR aynı zamanda günümüzde kullanılan birçok moleküler metodun temelini oluşturmaktadır. Reaksiyon her ne amaçla çalışılırsa çalışılsın, her gen bölgesi için, kullanılacak reagent ve PCR parametrelerinin optimizasyonunun yapılması gerekmektedir. Hatta yapılan optimizasyonun, PCR’ın gerçekleştirileceği farklı aletler ve laboratuvarlar arasında bile tekrar yapılması gerekebilmektedir. Bu derlemede, kısaca PCR’ın mikrobiyolojide kullanımı, PCR dizayn edilirken ve PCR’ın tüm aşamalarında kullanılan reagentler ve malzemelerde uyulması gereken ana standartlardan bahsedilecektir.

Anahtar Kelimeler:

Polimeraz zincir reaksiyonu (PCR), mikrobiyoloji, optimizasyon.

Polimeraz Zincir Reaksiyonu (PCR) Optimizasyonu

Polymerase chain reaction (PCR), a deoxyribonucleic acid (DNA) that lies between two known chain enzymatically amplify a specific DNA region as an in vitro technique becoming common everyday. PCR method, used for many purposes such as diagnosis, epidemiology and studies to determine the amount of DNA, are still under development. Microbiology is taking a significant proportion in PCR usage, like innovations of application in other fields. Furthermore, PCR is the fundamental molecular method that being used today for many purposes in detection of human and animal pathogenic microorganisms. It is needed to optimization of PCR parameters of each gene, regardless even what was targeted. PCR may be required to perform it again, even between different instruments and laboratories. In this review, PCR application in microbiology, the preperation of main standards must be followed at all stages of PCR reagents and materials when PCR a designed, will be mentioned.

Keywords:

Polymerase Chain Reaction (PCR), microbiology, optimization.,

PDF

___

Ahmed F.E., 2002. Detection of genetically modified organisms in foods. Trends in Biotechnology, 20, 215-223.
Aldemir O.S., Uçan U.S., 2001. Polimeraz zincir reaksiyonu (PZR), Temel prensipler. Hayvancılık Araştırma Dergisi, 1, 53-59.
Anonim, 2008. Avian Mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). Manual of Standards for Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter 2.3.5, Ofis International Epizootic terrestrial manual, Paris.
Anonim, 2009. Biotechnology in the diagnosis of infectious diseases and vaccine developments. Manual of Standards for Diagnostic Tests and Vaccines, Chapter 1.1.7. Office International Epizootics Terrestrial Manual, Paris.
Arı Ş., 2004. DNA’ nın polimeraz zincir reaksiyonu ile çoğaltılması. Editörler: Temizkan G., Arda N. Moleküler biyolojide kullanılan yöntemler. Biyogem yayınları, Nobel Tıp Kitabevleri, No:1, Bölüm 5, İstanbul, pp. 101-120.
Bilgehan H., 1992. Klinik mikrobiyoloji Tanı. Barış yayınları, Fakülteler Kitabevi, Ankara, pp. 56Caplin B.E., Rasmussen R., Bernard P.S., Wittwer C.T., 1999. The most direct way to monitor PCR amplification for quantification and mutation detection. Biochemica, 1, 5-8.
Carlı K.T., 2008. Hayvan İnfeksiyonlarında LightCycler PCR Kullanımı. Uludağ Üni Vet Med., 27, 1-10.
Cockerill F.R., Smith T.F., 2002. Rapid-cycle real-time PCR: A revolution for clinical microbiology. American Society for Microbiology News, 68, 77-83.
Cockerill F.R., 2003. Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Archieves of Pathology and Laboratory Medicine, 127, 1112-1120.
Çetinkaya B., 1998. Polimeraz zincir reaksiyonu (PCR) temel prensipler. Fırat Üniversitesi Sağlık Bilimleri Dergisi, 12, 149-156.
Elenitoba-Johnson O., David D., Crews N., Wittwer C.T., 2008. Plastic versus glass capillaries for rapid-cycle PCR. Biotechniques, 44, 4874
Greenfield L., White T.J., 1993. Sample preparation methods. Editörler: Persing D.H., Smith T.F.S., Tenover F.C., White T.J. Diagnostic molecular microbiology, 1 baskı. American society for microbiology, Washington, pp. 122-137.
He Q., Marjamaki M., Soini H., Mertsola J., Viljanen M.K., 1994. Primers are decisive for sensitivity of PCR. BioTechniques, 17, 82-87.
Howe C.J., Ward E.S., 1989. Nucleic acids sequencing. IRL Pres at Oxford University Pres, Oxford, page 53-114.
Innis M.A., Gelfand D.H., Sninsky J.J., 1995. Optimization of PCR’s. Editörler: Innis M.A., Gelfand D.H., Sninsky J.J. PCR Applications protocols for fanctional genomics. Academic pres, San Diego, pp. 39-67.
Klein D., 2002. Quantification using real-time PCR technology: Applications and limitations. Trends in Molecular Medicine, 8, 257-260.
Kleven S.H., Yoder H.W., 1984. Mycoplasmosis. Editörler: Purchase H.G., Arp L.H., Domermuth C.H., Pearson J.E. A laboratory manual for the isolation and identification of avian pathogens, 3th edition, American Association of Avian Pathologists, Kenet Square, Pennsylvania, pp. 57-62.
Kleven S., Jordan H.F.T.W., Bradbury J.M., 19 Avian Mycoplasmosis (Mycoplasma gallisepticum). Manual of Standards for Diagnostic Tests and Vaccines. Office International Des Epizootics, Paris, pp. 512-521. Kubista M., Andrade J.M., Bengtsson M., Forootan A., Jonak J., Lind K., 2006. The real-time polymerase chain reaction. Molecular Aspects in Medicine, 27, 95-125.
Lauerman L., Hoerr F.J., Sharpton A.R., Shah S.M., Santen V.L.V., 1993. Development and application of polymerase chain reaction assay for Mycoplasma synoviae. Avian Diseases, 37, 829-8
Lee D.S., Tsai C.Y., Yuan W.H., Chen P.H., 200 A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes. Microsystem Technologies, 10, 5795
Ley D.H., Avakian A.P., Berkhoff J.E., 1993. Clinical Mycoplasma gallisepticum infection in multiplier breeder and meat turkeys caused by F strain: identification by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, restriction endonuclease analysis and the polymerase chain reaction. Avian Diseases, 37, 854- 862.
Ley D.H., 2003. Mycoplasma gallisepticum infection. Editörler: Saif Y.M., Barnes H.J., Fadly A.M., Glisson J.R., Mcdougald L.R., Swayne D.E. Diseases of poultry, Iowa State University Press, 11. edition, pp. 722-743.
Lowe T., Sharefkin J., Yang S.Q., Diefenbach C.W., 1990. A computer program for selection of oligonucleotide primers for polymerase chain reactions. Nucleic Acids Research, 18, 1757-1761.
Marenda M.S., Sagne E., Poumarat F., Citti C., 200 Suppression substractive hybridization as a basis to assess Mycoplasma agalactia and Mycoplasma bovis genomic diversity and speciesspecific sequences. Microbiology, 151, 475-489. Mcpherson M.J, Hames B.D., Taylor G.R., 1995. A practical approach. Oxford Univresity Pres, Amerika, pp. 7-118.
Mhlanga M.M., Malmberg L., 2001. Using Molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods, 25, 463-4
Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H., 1986. Spesific enzymatic amplification of DNA in vitro: polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 51, 263-273.
Mullis K.B., Faloona F.A., 1987. Spesific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods of Enzimology, 155, 335-3
Persing D.H., 1993. Diagnostic molecular microbiology, principles and applications. Editörler: Persing D.H., Smith T.F., Tenover F.C., White T.J. American Society for Microbiology, Washington, pp. 88-104.
Roux K.H., 1995. Optimization and troubleshooting in PCR. Genome Research, 4, 185-194.
Rychlik W., Rhoads R.D., 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Research, 17, 8543-8549.
Sachse K., 2004. Specifity and performance of PCR detection assays for microbial pathogens. Molecular Biotechnology, 26, 61-79.
Saiki R.K., Scharf S., Faloona F., Mullis K.B., 19 Enzymatic amplification of B-Globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230, 1350-1354.
Saiki K.R., Gelfand H.D., Stoffi S., Scharf J.S., Higuchi R., Horn T.G., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491.
Saiki K.R., Walsh P.S., Leverson C.H., Erlich H.A., 1989. Genetic analysis of amplified DNA with immobilized sequence-spesific oligonucleotide probes. Proceedings of National Academic Sciences, USA, 86, 6230-6234.
Saiki K.R., Gelfand H.D., Stoffi S., Scharf J.S., Higuchi R., Horn T.G., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491.
Sarikaya A.T., 2004. DNA’nın izolasyonu ve analizi. Editörler: Temizkan G., Arda N. Moleküler biyolojide kullanılan yöntemler. Biyogem yayınları, Nobel Tıp Kitabevleri, No:1, Bölüm 2, İstanbul, pp. 55-80.
Sambrook J., Russell D.W., 2001. Molecular cloning, A laboratory manual. Third edition, Volume 2, chapter 8, New York.
Silveira R.M., Fiorentin L., Marques E.K., 1996. Polymerase chain reaction optimization for Mycoplasma gallisepticum and M. synoviae diagnosis. Avian Diseases, 40, 218-222.
Steffan R.J., Atlas R.M., 1991. Polymerase chain reaction: Applications in environmental microbiology. Annual Review of Microbiology, 45, 1371
Şahin F., Çiftçi M., Pirim İ., 2000. Polimeraz zincir reaksiyonu (PCR). II. Uygulamalı moleküler biyoloji teknikleri kurs notları. Ankara Üniversitesi Biyoteknoloji Uygulama ve Araştırma Merkezi, Teo I.A., Choi J.W., Morlese J., Taylor G., Shaunak S., 2002. LightCycler QPCR optimization for low copy number target DNA. Journal of Immunological Methods, 270, 119-133.
Wilson I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63, 3741-3751.
Wittwer C.T, Fillmore G.C, Hillyard D.R., 1989. Automated polymerase chain reaction in capillary tubes with hot air. Nucleic Acids Research, 17, 4353-4357.
Wittwer C.T., Fillmore G.C., Garling D.J., 1990. Minimazing the time required for DNA amplification by efficient heat transfer to small samples. Analytical Biochemistry, 186, 328-331.
Wittwer C.T., Garling D.J., 1990. Rapid cycle DNA amplification: Time and temperature optimization. Biotechniques, 10, 76-83.
Wittwer C.T., Rine K.M., Andrew R.V., David D.A., Gundry R.A., Balis U.J. 1997. The LightCycler: A microvolume multisample fluorimeter with rapid temperature control. Biotechniques, 22, 176-181.
Valasek M.A., Repa J.J., 2005. The power of real-time PCR. Advances in Physiology Education, 29, 151-159.