Murat Kemal AVCI, Erdem TEZCAN, Safiye AVCI, Haluk CAMCI

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

Lineer Poliakrilamid (LPA), kısa ve küçük moleküler ağırlıklı DNA moleküllerinin DNA izolasyonunda verimi arttırıcı ajan olarak kullanılan polimerlerden biridir. Kromatin imminopresipitasyon yöntemi DNA-Protein etkileşimlerinin aydınlatılmasında yaygın olarak kullanılan etkili ve verimli bir yöntemdir. Ardışık birçok aşamadan oluşan ChIP yönteminde kırpılmış DNA parçalarının tekrar izole edilmesi aşamasında DNA parçalarının boyutlarının küçük olmasına bağlı olarak kayıplar yaşanması önemli bir problem oluşturmaktadır. Bu çalışmada ülkemizde ve dünyada ticari değeri çok yüksek bir ökaryot organizma olan pamuk bitkisine yönelik uygulanan ChIP yönteminde kırpılmış küçük moleküler ağırlıklı DNA parçalarının daha yüksek verimle izole edilebilmesinde LPA matriksi kullanılmasının etkisi araştırılmıştır. Burada deneysel olarak ChIP yöntemi uyarlanarak ultrasonikasyon yöntemiyle DNA parçalama işlemi ve ardından parçalanmış DNA moleküllerinin izolasyonu aşamalarına kadar uygulanmıştır. İstatistiksel analizlerde ChIP deneyinin başlangıcında kullanılan DNA izolatlarının ve ultrasonikasyonla mekanik olarak parçalanmış DNA moleküllerinin Nanodrop cihazıyla ng/µl cinsinden konsantrasyonları karşılaştırılmıştır. Deneysel ve istatistiksel analizler, LPA uygulamasının DNA izolasyon etkinliğini 0,59 kat arttırdığını göstermiştir. Bu verilere dayanarak, pamuk bitkisine yönelik ChIP yönteminde LPA kullanılmasının deney başarısını doğrudan etkileyeceği ve çok daha verimli sonuçlar alınmasına katkı sağlayacağı ispatlanmıştır. Bu çalışma doğrudan pamuk bitkisine özel ChIP uygulamasında kırpılmış DNA izolasyonundaki verimi arttırmaya odaklanılması açısından özgündür.

Anahtar Kelimeler:

Lineer poliakrilamid, Kromatin immünopresipitasyonu, Pamuk, Kırpılmış DNA izolasyonu, Verimlilik

Application of Linear Polyacrylamide (LPA) Matrix in Cotton Chromatin Immunoprecipitation to Increase Sheared DNA Isolation Efficiency

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.

Keywords:

lineer polyacrylamide, chromatin immunoprecipitation, Cotton, Sheared DNA isolation, Efficiency,

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