Concentration 50 gm L OD d NTPs A
- Slides: 46
Concentration 50 g/m. L OD d. NTPs A 260 = 1. 60 S. S DNA A 260 = 1. 37 D. S DNA A 260 = 1 1. 37 1. 185 1. 0 Tm = OD增加值的中点温度(一般为 85 -95℃) ℃
Marmur-Doty Formula 1 x SSC ( 0. 15 M Nacl + 0. 015 M Sodium Limonate ) GC% = 30 70% OD Tm = 69. 3 + 0. 41 × GC% 1. 37 what is meaning ? 1. 185 Evaluation GC% of DNA ℃ 1. 0 Tm 1 < Tm 2
DNA 分子的复性 (anneal or renaturation) Denaturation ▲ D. S DNA S. S DNA ▼ Renaturation 复性过程依赖于单链分子间的随机碰撞 ( Depends on the collision of complementary S. S. DNA )
d. Ct / dt = -KCt 2 积分 Ct / C 0 = 1 / 1+KC 0 t 当 Ct / C 0 = 1/2 时 Ct / C 0 = 1/2 = 1 / 1+ KC 0 t(1/2) K = 1 / Cot(1/2) = 1/K (mol. Sec / L)
杂交检测 D. S. DNA → S. S. DNA × S. S. DNA ( as probe) p 32 or biotin labeled Xfilm Column absorb D. S DNA
1975 E. M. Southern blot S. S. DNA × S. S. DNA 1977. J. C. Alwine Northern blot S. S. RNA × S. S. DNA 1978. Hany Towbin Western blot Protein × antibody * 原位分子杂交 (In situ hybridization )
Southern Blotting Western Blotting Northern Blotting
1. 历史 1972. Herbert Boyer 限制性核酸内切酶II (RE-II) 1975. Sanger C. 1976. “+ , —” 法 1977. Maxam Gilbert 1978. 化学定序法 1977. Sanger C. 1978. 酶)法 dd. NTP(
Chemical sequencing G系统 脱G A+G系统 脱嘌呤 C系统 脱C T + C系统 脱嘧啶 A A C A G C TT C A
(2) 双脱氧法 N dd. ATP dd. CTP dd. TTP H dd. GTP H 4 dd. NTP系统中 DNA链均终止 在dd. NTP(二脱 氧核苷酸) A C T G C T G A C T T C G A C A A
六 DNA的甲基化(Methylation) (m 5 C in Eukaryote)
1. DNA中m 5 C的特点 CH 3 H 5 m 5 C H HNH 4 6 3 N 2 1 O N ● 对Msp. I, Hpa. II 位点的高频修饰 Msp. I Cm. C GG C Cm. GG Hpa. II +
● m 5 C 与基因突变的关系 C → m 5 C → T → gene mutation (癌变) HNH CH 3 5 O CH 3 5 5 氧化脱氨 O O O C m 5 C T G G A
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