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ชื่อเรื่อง : ผลของ RAPTA-EA1 ต่อการเกิดดีเอ็นเอแอดดัค การสังเคราะห์ดีเอ็นเอ การซ่อมแซมดีเอ็นเอ และการกระตุ้นการถอดรหัสของยีนกดมะเร็งบีอาร์ซีเอวัน
นักวิจัย : อดิศร รัตนพันธ์
คำค้น : RAPTA-EA1 , Ruthenium , BRCA1 , Adduct formation , DNA amplification , DNA damage and repair , Transcriptional activation
หน่วยงาน : คณะเภสัชศาสตร์
ผู้ร่วมงาน : -
ปีพิมพ์ : 2557
อ้างอิง : -
ที่มา : -
ความเชี่ยวชาญ : -
ความสัมพันธ์ : -
ขอบเขตของเนื้อหา : -
บทคัดย่อ/คำอธิบาย :

9. Methodology

 

9.1. Materials

 

The ruthenium complex, RAPTA-EA1, used in this study is generously provided by Prof. Dr. Paul J. Dyson, Director of the Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology, Lausanne, Switzerland (Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, EPFL-BCH, CH-1015 Lausanne, Switzerland). Taq DNA polymerase, dNTP and Tris–HCl, are purchased from New England Biolabs. Other reagents are of highest purity grade.

 

9.2. Biophysical characterization of RAPTA-EA1 binding to plasmid DNA  

       and BRCA1 fragment  

 

9.2.1. Conformational study of the in vitro ruthenation of plasmid pBIND  

          DNA

 

Agarose gel electrophoresis is used to probe the ruthenium complex-mediated conformational changes of plasmid DNA using the plasmid pBIND DNA as a model. Plasmid pBIND DNA (6360 bp) (Promega, USA) (Fig. 3) is transformed into Escherichia coli DH5a. Transformed E. coli cells are grown in Luria broth medium with 50 mg/ml ampicillin at 37 ºC for 16 h. The cells are harvested and lyzed. The plasmid DNA is purified as described previously [43]. Samples of pBIND plasmid (4 μg) are incubated with RAPTA-EA1 in 20 μl of double distilled water at various molar ratios of ruthenium per DNA nucleotide (rb) at 37 ºC for 24 h in the dark. The Ru-treated DNA is precipitated with absolute ethanol and centrifuged at 13,500x g for 30 min at 4 ºC. After drying, the DNA pellet is washed with 70% ethanol, dried in vacuum, and redissolved in 20 μl of double distilled water. DNA adducts are electrophoresed on a 1% agarose gel. The gel is stained with ethidium bromide and visualized under UV light.

It is expected that the mobility of the metal complex-treated plasmid DNA is reduced as the molar ratio of ruthenium per DNA nucleotide increases. This observation can be explained by changes in plasmid supercoiling; supercoiling reduces the three dimensional size of the DNA plasmid such that the rate of DNA plasmid migration during electrophoresis decreases as the number of supercoils decreases.

 

 

 

Fig. 3. Vector circle map and multicloning site of pBIND (Promega, USA)

 

 

 

 

9.2.2. Conformational study of the in vitro ruthenation of the BRCA1

          fragment  

 

9.2.2.1. Preparation of the 696 base pair (bp) BRCA1 fragment

 

Messenger RNA (mRNA) is extracted from white blood cells using an mRNA isolation kit and biotinylated oligo-dT (Qiagen). The purified mRNA is used for complementary DNA (cDNA) synthesis and the amplification of the 696-bp BRCA1 fragment (nucleotide 4897–5592) is performed with the Qiagen OneStep RT-PCR Kit (Qiagen). The RT-PCR mixture is prepared in a 1.5 ml microcentrifuge tube with a final volume of 50 μl, containing 5 μl reaction buffer,    400 μM of each dNTP, 0.6 μM of forward primer                                              (5/-AGCAGGGAGAAGCCAGAATTG-3/), 0.6 μM of reverse primer (5/-TCAGTAGTGGCTGTGGGGGAT-3/), OneStep RT-PCR enzyme mix (Qiagen), and RNase-free water. The template RNA is finally added in order to initiate the PCR reaction using two-step thermal cycling. The first step comprises one cycle at 48 ºC for 45 min, allowing the synthesis of the first strand cDNA by the action of reverse transcriptase. The reverse transcriptase is inactivated at 94 ºC for 2 min. The second step includes 40 cycles of denaturation at 94 ºC for 30 s, annealing at 60 ºบรรณานุกรม

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