Pamukta Kromatin İmmünopresipitasyon Yönteminde Kırpılmış DNA İzolasyon Verimini Arttırmak için Lineer Poliakrilamid (LPA) Matriksinin Uygulanması

Abstract

Linear Polyacrylamide (LPA) is one of the polymers used as a yield-enhancing agent in DNA isolation of short and small molecular weight DNA molecules. The Chromatin immunoprecipitation (ChIP) method is widely used to elucidate DNA-Protein interactions. In ChIP, which consists of many successive steps, it is an important problem to experience losses due to the small size of the DNA fragments during the re-isolation of the sheared DNA fragments. In this study, the effect of using LPA matrix on the isolation of small molecular weight DNA fragments with higher efficiency in the ChIP method applied to cotton plant, which is a eukaryotic organism with high commercial value in our country and in the world, was investigated. Here, the ChIP method was adapted and applied till the DNA fragmentation process by ultrasonication and the isolation of the fragmented DNA molecules. In statistical analyzes, the concentrations of DNA isolates used at the beginning of the ChIP experiment and the DNA molecules mechanically fragmented by ultrasonication, measured by the Nanodrop, were compared in ng/µl. Experimental and statistical analyzes showed that LPA application increased DNA isolation efficiency by 0.59 times. Based on these data, it has been proven that using LPA in ChIP method with cotton will directly affect the success of the experiment and contribute to get much more efficient results. This study is unique in that it focuses on improving the yield of sheared DNA isolation in direct cotton plant-specific ChIP application.