Silindrospermopsin genleri için Dâhili çoğaltma kontrollerinin (Dçk) tasarımı ve sınırlayıcı parça uzunluk çeşitliliği (Spuç) testinin geliştirilmesi

Çevre örneklerinden elde edilen DNA sonraki moleküler işlemleri engelleyen yabancı maddeler içerebilir. Dâhili çoğaltma kontrollerinin (DÇK) kullanımı Polimeraz Zincirleme Reaksiyonu (PCR)’unu engelleyen bu maddeleri tespit etmenin yollarından biridir. Çalışmada, bu çoğaltma kontrollerinin en yaygın kullanılan silindrospermopsin genleri cyrB ve cyrC için kullanılması konusunda bir yöntem geliştirilmiştir. DÇK’ların kullanımı ile A.B.D’nin Florida eyaletinde bulunan Dora, Griffin, Crescent, Buffalo Bluff ve George Gölleri’nden toplanan örneklere ait DNA’ların PCR’ı engellediği gösterilmiştir. DNA’ların engelleyici maddelerden temizlenmesi sonrası Crescent, Buffalo Bluff ve George Gölleri’nde cyrB ve cyrC genleri tespit edilmiştir. Göl örnekleri, Aphanizomenon ovalisporum ve Cylindrospermopsis raciborskii’ye ait cyrB genleri için geliştirilen Sınırlayıcı Parça Uzunluk Çeşitliliği (SPUÇ) analizi bu örneklerde silindrospermopsin üreten siyanobakterinin A. ovalisporum olduğunu göstermiştir.

Anahtar Kelimeler:

genler, polimeraz zincir reaksiyonu, mavi-yeşil bakteriler, DNA, kısıtlı fragment uzunluğu polimorfizmi

Design and testing of internal amplification controls (IAC) and development of a restriction fragment length polymorphism (RFLP) assay for Cylindrospermopsin genes.

Environmental DNA may contain impurities that inhibit downstream molecular applications. Use of an internal amplification control (IAC) is one way to detect inhibition of the polymerase chain reaction. We describe a strategy to design these amplification controls for the most commonly tested cylindrospermopsin genes cyrB and cyrC. We demonstrate PCR inhibition from DNA of samples collected from lakes Dora, Griffin, Crescent, Buffalo Bluff and George in Florida, USA by use of these internal controls. Further cleaning of DNA from impurities resulted in detection of cyrB and cyrC genes in samples from lakes Crescent, Buffalo Bluff and George. Comparison of restriction digests of cyrB fragments from lake samples, Aphanizomenon ovalisporum and Cylindrospermopsis raciborskii suggested the former cyanobacterium as the likely source of cylindrospermopsin in these samples.

Keywords:

genes, polymerase chain reaction, cyanobacteria, DNA, restriction fragment length polymorphism,

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