Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

Kandan DNA izolasyonu, nDNA ve mtDNA’yı elde etmek için yaygın kullanılan bir uygulamadır. Donmuş çözünmüş kanın (DÇK) santrifüjü sonrası elde edilen çökeltiden DNA izolasyonun gerçekleştirilebileceği daha önce gösterilmişti ve bu ön uygulama DNA izolasyonu üzerine olumlu sonuçlara sahipti. Fakat, bu ön uygulama için hangi santrifüj değerlerinin kullanılabileceği ve bunların etkileri bilinmemektedir. Bu çalışmanın amacı belirtilen ön uygulama için uygun santrifüj değerlerini belirlemek ve bu santrifüj değerlerinin izole edilen DNA üzerine ektilerini göstermekti. Bu amaçla, DÇK’nin farklı değerlerde santrifüjüyle elde edilen çökelti ve süpernatantdan DNA izolasyonları gerçekleştirildi. Sonra, izole edilen DNA örneklerinden spektrofotometrik, jel elektroforezi ve gerçek-zamanlı PCR analizleri gerçekleştirildi. Sonuç olarak, DÇK’nin, 5.000×g’de 2 dakika ya da üzeri değerde santrifüjü genetik materyali tamamen çöktürmeyi sağladı. Bu durum ayrıca yüksek miktarda DNA elde edilmesine imkan sağladı. Belirtilen santrifüj seviyelerinde elde edilen çökeltiden izole edilen DNA örneklerinde mtDNA/nDNA oranı değişmedi fakat, DNA bütünlüğü azaldı. Sonuç olarak, DÇK’nin 5.000×g’de 2 dakika ya da üzerinde santrifüjü, DNA izolasyonundan önce DÇK’de bulunan genetik materyali çöktürmek ve yıkamak için kullanılabilir.

Anahtar Kelimeler:

DNA izolasyonu, Donmuş-çözünmüş kan, Kan, Santrifüj

Effects of Centrifugation at Different Levels of Freeze-Thawed Blood on DNA Isolation

DNA isolation from blood is a commonly used application to obtain nDNA and mtDNA. It was previously shown that DNA isolation could be performed from the pellet obtained after centrifugation of freeze-thawed blood (FTB), and this pretreatment had constructive results on DNA isolation. However, which centrifugation levels can be used for this pretreatment, and their effects are unknown. The aim of the study was to determine appropriate centrifugation levels for this pretreatment and show their effects on isolated DNA. For this purpose, DNA isolations were carried out from pellet and supernatant obtained by centrifugation at different levels of FTB. Then, spectrophotometric, gel electrophoresis, and real-time PCR analyses were performed in the isolated DNA samples. As a result, centrifugation of FTB at 5,000×g for 2 min or over let genetic material to pellet completely. This also caused to obtain high amount of DNA. mtDNA/nDNA ratios did not change in the isolated DNA samples from pellets obtained by defined centrifugation levels, but the DNA integrity decreased. To conclude, centrifugation of FTB at 5,000×g for 2 min or over can be used to harvest and wash genetic material found in FTB before DNA isolations.

Keywords:

Blood, Centrifuge, DNA isolation, Freeze-thawed blood,

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