分子生物学-ch-2-The-nature-of-DNA.pptVIP

Stability of secondary structure is stabilized by   hydrogen bonds  Base Stack (including  hydrophobic  forces and Wan de wal)    SYNERGY On the other hand, a repulsive force ,namely, that between the negatively charged phosphate groups in complementary strands. Nucleic acid bases absorb 260-nm light strongly, and the amount of light absorbed by a given number of bases depends on their proximity to one another. 2.3.3 Tm http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html Hyperchromie: If a solution of double-stranded DNA has a value of A260 = 1.00, a solution of single-stranded DNA at the same concentration has a value of A260 = 1.37. the corresponding value for free bases is 1.60. hyperchromicity of a DNA : The UV absorption is increased when DNA duplex is denaturated. If a DNA solution is slowly heated and the A260 is measured at various temperatures, a melting curve such as that shown in Figure 3-9 is obtained. So that absorbance can be used to measure denaturation or degradation of nucleic acids. The rise in A260 occurs over a relatively narrow range of 6—8℃. maximum A260 is about 37% higher than the starting value. Tm： the melting temperature ，the temperature at which the rise in A260 is half complete. Tm increases with percent G + C, a higher temperature (more energy) is required to disrupt a G- C pair than an A-T pair. reagents that increase the solubility of the bases and therefore decrease interactions, such as base stacking, also lower Tm. 引物长度,碱基组成,引物使用缓冲的离子强度有关. 长度为25mer以下的引物,Tm计算公式为：Tm = 4℃(G + C)+ 2℃(A + T) 对于更长的寡聚核苷酸,Tm计算公式为： Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) – 600/size 公式中,Size = 引物长度. DNA二级结构是碱不稳定的！ 2.3.3 renaturation Interpretation: two different classes of fragments 1) fast renaturing - repetitive fragments ~15% of the DNA ; 2) slow