Bazı kanser hücrelerinden nükleik asitlerin izolasyonu ve analizi
Isolation and analysis of nucleic from some cancer cells
Yazar: REYHAN ÇOLAK VERİMLİ
Danışman: PROF.DR. CEVAT AYVALI
Ankara Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Doktora, 1996
Konu: Biyoloji = Biology
Dizin: DNA = DNA ; Neoplazm hücreleri = Neoplastic cells ; Nükleik asit = Nucleic acids ; RNA = RNA ; Yalıtım = Insulation

Özet:
İÜ ABSTRACT Ph.D. Thesis Isolation and Analysis of Nucleic Acids from Some Cancer Cells Reyhan VERÎMLÎ Ankara University Graduate School of Natural and Applied Sciences Department of Biology Supervisor: Prof. Dr. Cevat AYVALI 1996, Page: 76 Jury: Prof. Dr. Cevat AYVALI Prof. Dr. Ayşe AYHAN Assoc. Prof. Dr. Kevser PÎŞKÎN The aim of this study is to obtain sufficiently and optimally purified nucleic acids from cancer cells and tissues and to image them by Scanning Tunneling Microscopy (STM). DNA and RNA are obtained from haemato logic and solid tumours along with the cultured cells by two distinct methods. The cytoplasmic RNA was isolated from blood and the cultured cells by the NP-40 (Nonidet P-40) method, also isolation of the total RNA from both cells and tissues were performed by the GTC (Guanidinium Thio Cyanate) method. The isolation of DNA from nuclear pellet following the isolation of RNA by the method NP-40 was attempted in this study for the first time. In comparison with the standard method, it was possible to isolate DNA by this modified method more efficiently. Furthermore, DNA and RNA could be isolated from the same material. The quantity of the isolated nucleic acids were examined spectrophotometrically and the quality was analysed electrophoretically.iv Using modified DNA isolation method, 17.1 ug/ml and 42.9 ug/ml of DNA in average were obtained from the normal blood samples and cancerous blood samples, respectively. By the standard method, 2.3-3.2 ug/mg DNA was obtained from normal tissue samples and 5.7 ug/mg DNA from gastrointestinal tumour biopsies. The purity of the isolated DNA were found to be in the range of 1.3-1.8 by the standard method and 1.5-2.0 by the modified method. The sufficient amount of RNA was obtained by NP-40 and GTC RNA isolation methods and their purity was found to be 1.5-2.2 by both methods. The double helix structure, denatured form, restriction enzyme cleaved DNA and cytoplasmic, RNA were imaged by STM. In the analysis of cytoplasmic RNA, the images are found to be associated with rRNA, however it was determined that this was not consistent with the known- prokaryotic 16S rRNA. But we were able to determine tRNA molecules in the RNA samples obtained from cancer cells and were imaged in this study for the first time. As a result of this study, the purifying process which formes the first step of nucleic acid research was optimized and the types of DNA and RNA were clearly imaged by STM. It is concluded that may not only contribute to the structural aspects of nucleic acids but may also help explaining their biochemical aspects such as behaviour in various media, affinities to ions and other molecules and may be utilized in the determination of genetic disorders by sequence analysis using specific probes. KEY WORDS: Cancer, DNA, RNA, isolation, Scanning Tunneling Microscope (STM).

Abstract:
İÜ ABSTRACT Ph.D. Thesis Isolation and Analysis of Nucleic Acids from Some Cancer Cells Reyhan VERÎMLÎ Ankara University Graduate School of Natural and Applied Sciences Department of Biology Supervisor: Prof. Dr. Cevat AYVALI 1996, Page: 76 Jury: Prof. Dr. Cevat AYVALI Prof. Dr. Ayşe AYHAN Assoc. Prof. Dr. Kevser PÎŞKÎN The aim of this study is to obtain sufficiently and optimally purified nucleic acids from cancer cells and tissues and to image them by Scanning Tunneling Microscopy (STM). DNA and RNA are obtained from haemato logic and solid tumours along with the cultured cells by two distinct methods. The cytoplasmic RNA was isolated from blood and the cultured cells by the NP-40 (Nonidet P-40) method, also isolation of the total RNA from both cells and tissues were performed by the GTC (Guanidinium Thio Cyanate) method. The isolation of DNA from nuclear pellet following the isolation of RNA by the method NP-40 was attempted in this study for the first time. In comparison with the standard method, it was possible to isolate DNA by this modified method more efficiently. Furthermore, DNA and RNA could be isolated from the same material. The quantity of the isolated nucleic acids were examined spectrophotometrically and the quality was analysed electrophoretically.iv Using modified DNA isolation method, 17.1 ug/ml and 42.9 ug/ml of DNA in average were obtained from the normal blood samples and cancerous blood samples, respectively. By the standard method, 2.3-3.2 ug/mg DNA was obtained from normal tissue samples and 5.7 ug/mg DNA from gastrointestinal tumour biopsies. The purity of the isolated DNA were found to be in the range of 1.3-1.8 by the standard method and 1.5-2.0 by the modified method. The sufficient amount of RNA was obtained by NP-40 and GTC RNA isolation methods and their purity was found to be 1.5-2.2 by both methods. The double helix structure, denatured form, restriction enzyme cleaved DNA and cytoplasmic, RNA were imaged by STM. In the analysis of cytoplasmic RNA, the images are found to be associated with rRNA, however it was determined that this was not consistent with the known- prokaryotic 16S rRNA. But we were able to determine tRNA molecules in the RNA samples obtained from cancer cells and were imaged in this study for the first time. As a result of this study, the purifying process which formes the first step of nucleic acid research was optimized and the types of DNA and RNA were clearly imaged by STM. It is concluded that may not only contribute to the structural aspects of nucleic acids but may also help explaining their biochemical aspects such as behaviour in various media, affinities to ions and other molecules and may be utilized in the determination of genetic disorders by sequence analysis using specific probes. KEY WORDS: Cancer, DNA, RNA, isolation, Scanning Tunneling Microscope (STM).