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Chapter 5

Chapter 5. Molecular Biology-DNA. 5-1 Introduction – Gene History. 1865 Mendel: The basic unit of inheritance is a gene . Mendel’s work was forgotten until 1900s. 1944 The gene was known to be made of DNA ( D eoxyribo n ucleic A cid) .

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Chapter 5

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  1. Chapter 5 Molecular Biology-DNA 1-

  2. 5-1 Introduction – Gene History • 1865 Mendel: The basic unit of inheritance is a gene. • Mendel’s work was forgotten until 1900s. • 1944 The gene was known to be made of DNA (Deoxyribonucleic Acid). • 1953 James Watson and Francis Crick : Double helical structure of DNA. 1-

  3. 動物細胞 • DNA位於細胞核內之「核仁」 1-

  4. 5-2 DNA 的構造 Double Helix 1-

  5. DNA的成分與化學鍵結： 1-

  6. 核甘酸 • 核甘酸(Nucleotide)為核酸分子構成單元 • 核甘酸包含： • 五碳糖、磷酸基、含氮鹼基 1-

  7. 核甘酸構成 DNA and RNA • Nucleotide (核甘酸)的含氮鹼基有五種： adenine、guanine、cytosine、thymine、uracil • DNA(deoxyribonucleic acid , 去氧核糖核酸) {A, G, C, } • RNA(ribonucleic acid, 核糖核酸) {A, G, C, } 1-

  8. DNA四種含氮鹼基 1-

  9. DNA Length • The total length of the human DNA is about 3109(30億) base pairs. • 1% ~ 1.5% of DNA sequence is useful. • # of human genes: 30,000~40,000 • Genes: 1-

  10. Base Pairs： G≡C、A＝T 1-

  11. 5-3 DNA 定序 (1) 打破細胞 (2) 加入乙醇 (3) 以玻棒撈取 DNA (4) 乾燥後溶出 DNA (5) 以限制脢切開 DNA (6) 以電泳檢視 1- Pharmacia

  12. 5-3-1 DNA 剪斷與連接 • Given DNA sequence: TGCACTTGAACGCATGCT Cut the sequence after random A: ATGCT length= ACGCATGCT length= AACGCATGCT length= ACTTGAACGCATGCT length= 1-

  13. Restriction Enzyme CIVIC, Madam AATTC G GAATTC AATTC G GAATTC G AATTC AATTC G Get An Apple To The Class Arber, Nathans, Smith (1978) Sticky Ends (Cohesive Ends) EcoRI 1- Juang RH (2004) BCbasics

  14. 以限制脢及連結脢進行核酸剪接 GAATTC GAATTC CTTAAG CTTAAG G G G G G AATTC AATTC AATTC AATTC CTTAA CTTAA CTTAA CTTAA CTTAA G G G G AATTC G EcoRI EcoRI sticky end EcoRI sticky end DNA Ligase 1- Juang RH (2004) BCbasics

  15. 核酸的 Hairpin 或 Cruciform 構造 1- Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.339

  16. 5-3-2 電泳分離法 1-

  17. 做分離鹼基電泳的方法 Maxam-Gilbert's Method: ATCGATCG ATCGATCGAT ATCG Sanger's Method: Analogue 32P 32P 32P 32P 32P A,T,C,G A ATCG ATCGA ATCG TAGCTAGCTA TAGCTAGCTA TAGCTAGCTA 化學法 AT ATCGAT Specific Reaction to G 生合成法 or Template A 1- Juang RH (2004) BCbasics

  18. 生合成核酸定序法的反應 P R P R P R P R P R P R 1 5’ 1 A 2 3’ OH 5’ 3 A 2- 4 PO4 2 5 3’ H H 6 5’ ddNTP 中止 Phosphodiester bond dideoxynuceotide 3’ 雙去氧衍生物 1- Juang RH (2004) BCbasics

  19. DNA 的限制脢圖譜檢定 8 kb 2 kb A 7 kb 3 kb B 5 kb 3 kb 2 kb A + B Restriction enzymes A B? 10 kb CK A B A+B M 1- Juang RH (2004) BCbasics

  20. ATCGATCGAT 32P ATCGATCGA 32P ATCGATCG 32P ATCGATC 32P ATCGAT 32P ATCGA 32P G ATCG 32P C ATC 32P T AT 32P A 32P A 5-3-3 DNA 定序法 膠體電泳可以分開各種不同長度 的核酸，在電泳膠片依序排開︰ Polyacrylamide Gel Electrophoresis 隨便切，各片段都被放射性元素標記，顯示在底片上但這樣的圖譜不能判別出各核酸端點的核苷酸種類 若能把尾端核苷酸相同的片段提 出(分別標記)，跑在同一行，則 比較這樣的四行，即可讀出序列 1- Juang RH (2004) BCbasics

  21. 練習： T A G C 已知一段DNA序列如下， 請預測四條電泳圖譜 TACTATGCGC 1-

  22. mRNA 質 體 質體可進出宿主細胞 抗藥酵素 水解抗生素 抗藥性新菌種 非抗藥性菌種 5-4 基因工程5-4-1 基因重組的應用 抗藥性菌種 1-

  23. 目標基因殖入質體並轉形宿主菌 目標基因 染色體 宿主菌 重組質體 轉型宿主 目標基因 殖入質體 1 plasmid 1 cell 1- Juang RH (2004) BCbasics

  24. 目標基因的放大與篩選 培養皿 塗佈 1 cell line, 1 colony 挑出單一菌落 含有目標質體 1- Juang RH (2004) BCbasics

  25. PCR基因放大連鎖反應 染色体 PCR 可以把指定的基因片段數量放大 ↓ 生物親緣鑑定 1- Juang RH (2004) BCbasics

  26. Repeat Polymerase Chain Reaction(聚合脢連鎖反應) Denaturation- 95ºC Annealing- 55ºC Chain Extension- 72 ºC 1-

  27. CAG GTC CGG Val CAG GCC GCC CAG Thr GCC CAG CAG GTC 5-4-2 定點突變 Wild type (5) translation (1) Wild type protein + primer Mutant (2) primer translation (6) + polymerase (3) Mutant protein (4) Juang RH (2004) BCbasics replication 1-

  28. Original gene 原始 probe 5-4-3 RFLP 限制脢圖譜多形性 用來檢定兩種生物或個體在分子演化上的親疏關係 Gene insertion/duplication 插入或重複 Single base mutation 單一鹼基變性 Restriction enzyme digestion Electrophoresis 1- Juang RH (2004) BCbasics

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