() (Top) Effect of pH on circularization of linear oligodeoxyribonucleotide HT2

2011-12-31T00:01:35Z (GMT) by Jianying Qi Richard H. Shafer
<p><b>Copyright information:</b></p><p>Taken from "Covalent ligation studies on the human telomere quadruplex"</p><p>Nucleic Acids Research 2005;33(10):3185-3192.</p><p>Published online 2 Jun 2005</p><p>PMCID:PMC1142406.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Covalent ligation reactions were performed as described in Materials and Methods. The circularization reaction proceeded at pH 4.5 (lane 2), 5.0 (lane 3), 5.5 (lane 4), 6.0 (lane 5), 6.5 (lane 6) and 7.0 (lane 7), respectively. Lane 1: same reaction as the one loaded in lane 4, except for the absence of -cyanoimidazole. Lane 10: the 20mer d(T) as a molecular weight marker. (Bottom) Hydrolysis of HT2 ligation product by exonuclease VII. Lane 1: linear HT2 alone. Lane 2: linear HT2 after treatment with 20 U of exonuclease VII at 37°C for 2 h. Lane 3: ligation reaction mixture alone. Lane 4: ligation reaction mixture after treatment with 20 U of exonuclease VII at 37°C for 2 h. ( (Top) Loop size and phosphate group number dependency of the covalent ligation of human telomere sequences. Same reaction as the one loaded in lane 4 in , except for replacing sequence HT2 with HT1, d(pAGTAGTAGTAG), HT3, d(pAGTAGTAGTAGT), HT4, d(pAGTAGTAGTAGTp), HT5, d(pTAGTAGTAGTAGT) or HT6, d(pTAGTAGTAGTAGTp), respectively. Lane 1: HT1 alone. Lanes 2–7 contain reaction mixtures of HT1 (lane 2), HT2 (lane 3), HT3 (lane 4), HT4 (lane 5), HT5 (lane 6) and HT6 (lane 7), respectively. (Bottom) Hydrolysis of ligation product of HT4 and HT6 by exonuclease VII. Lane 1: HT4 alone. Lane 2: ligation reaction mixture of HT4. Lane 3: ligation reaction mixture of HT4 after treatment with 20 U of exonuclease VII at 37°C for 2 h. Lane 4: HT6 alone. Lane 5: ligation reaction mixture of HT6. Lane 6: ligation reaction mixture of HT6 after treatment with 20 U of exonuclease VII at 37°C for 2 h. () (Top) End position dependency of the covalent ligation of human telomeres. Same reaction as the one loaded in lane 4 in , top, except for replacing sequence HT2 with HT5, d(pTAGTAGTAGTAGT), HT6, d(pTAGTAGTAGTAGTp), HT7, d(pGTAGTAGTAGTAG) and HT8, d(pGTAGTAGTAGTAGp), respectively. Lanes 1, 3, 5 and 7: same reaction as the one loaded in lane 4, except for the absence of -cyanoimidazole. Lanes 2, 4, 6 and 8 contain reaction mixtures of HT5, HT6, HT7 and HT8, respectively. Lane 9: the 20mer of 5′-d(T)-3′ as a molecular weight marker. (Bottom) Hydrolysis of ligation product of HT6 and HT8 by exonuclease VII. Lane 1: HT6 alone. Lane 2: ligation reaction mixture of HT6. Lane 3: ligation reaction mixture of HT6 after treatment with 20 U of exonuclease VII at 37°C for 2 h. Lane 4: HT8 alone. Lane 5: ligation reaction mixture of HT8. Lane 6: ligation reaction mixture of HT8 after treatment with 20 U of exonuclease VII at 37°C for 2 h.