ddG ddA ddT ddC 测序反应 电 泳 负极 AACGTGGACT◆3' 30 AACGTGGAC AACGTGGA AACGTGG AACGTG AACGT 5432 AACG AAC AA 1 二 A 正极 5
26 生物化学与分子生物学教研室
S”-CTGACTTCGACAAAGAA-3'未知序列的单链DNA TGTTo 放射性标记的引物 Klenow酶 dNTP 双脱氧 ddATP ddCTP ddTTP ddGTP 了■顺 末端终 CTGACTTCGACAA ddG ddG 止法 ddG 反应混合物 ddGL。 凝胶电泳 3'1 5 较大片段 G 读出模板互 读出待测 补序列 序列 -D G A 较小片段 3 2
27 双脱氧 末端终 止法 读出模板互 补序列 dNTP 凝胶电泳 较大片段 较小片段 ddATP ddCTP ddTTP ddGTP 反应混合物 Klenow酶 未知序列的单链DNA 读出待测 序列 5´ CTGACTTCGACAAAGAA 3´ TGTT 放射性标记的引物 A C T G ddG ddG ddG ddG G A C T G A A G C T G T T 3´ 5´ C T G A C T T C G A C A A 5´ 生物化学与分子生物学教研室 3´
B.Sequencing of DNA ⊙5 GATC Protein Synthetic primer Single-stranded preparation sequence Hybridization DNA ⑥5 sequence © Phe 可 A 可 可 +4 dNTP +4 dNTP +4dNTP +4 dNTP +ddGTP ddATP +ddTTP +ddCTP 啊编 Polymerase Polymerase Polymerase Polymerase G A T C o Example:G 。 回=dd6 ⊙ d)Gel electrophoresis eVisualization of the fragments f Reading of(◆）：ACGATG 1.Method ④ 2.Sequence pattern
28 生物化学与分子生物学教研室
The Nobel Prize in Chemistry 1980 for his fundamental studies "for their contributions concerning of the biochemistry of nucleic the determination of base sequences acids,with particular regard in nucleic acids" to recombinant-DNA" Paul Berg Walter Gilbert Frederick Sanger 1/2 of the prize 1/4 of the prize 1/4 of the prize Harvard University, MRC Laboratory of Stanford University Biological Laboratories Molecular Biology Stanford,CA,USA Cambridge,MA,USA Cambridge, United Kingdom 29
29 The Nobel Prize in Chemistry 1980 "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA" "for their contributions concerning the determination of base sequences in nucleic acids" Paul Berg Walter Gilbert Frederick Sanger 1/2 of the prize 1/4 of the prize 1/4 of the prize Stanford University Stanford, CA, USA Harvard University, Biological Laboratories Cambridge, MA, USA MRC Laboratory of Molecular Biology Cambridge, 生物化学与分子生物学教研室 United Kingdom
Primer Template of 3 unknown sequence Dye-labeled four dNTPs four ddNTPs DNA自动测序法 Dye-labeled segments are applied to a DNA d apilla 设计原理：以Sanger法为基础 electrophoresis. 荧光标记物：不同颜色的荧光 分别代表A、G、C、T Laser beam Detector 电泳一电信号一显示 CCTGT TTCAT CGTGGT TCCGAAAT Computer-generated re sult after bands migrate past detector 30
30 DNA自动测序法 设计原理：以Sanger法为基础 荧光标记物：不同颜色的荧光 分别代表A、G、C、T 电泳 电信号 显示 生物化学与分子生物学教研室