Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

Abstract

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 both 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, which enable to obtain higher DNA yield. Real-time PCR results showed that mtDNA/nDNA ratios did not change in the isolated DNA samples which were performed from pellets obtained by defined centrifugation levels whereas isolated DNA integrity decreased. To conclude, centrifugation at 5 000 ×g for 2 min or over can be used to harvest and wash genetic material found in FTB before DNA isolations.