Gene Cloning & DNA Analysis

Transcription

1 CSS451 CSS/HRT 451 Gene Cloning & DNA Analysis Chapter 4-5 T-DNA LB auxin cytokin opine Oncogenic genes RB vir genes ori opine catabolism Guo-qing Song

2 Part 1 Basic principles Gene Cloning & DNA Analysis Part 2 Applications in Research Part 3 Applications in Biotechnology

3 The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) The range f DNA 4. Modifying enzymes P54-86

4 Exonucleases One at a time at the end of DNA molecule Endonucleases Break internal phosphodiester bonds within a DNA molecule P54-86

5 (a) An exonuclease (b) An endonuclease The reactions catalysed by the two different kinds of nuclease Figure 4.1 (P56) P54-86

6 (a) Bal31 The reactions catalysed by different types of exonuclease (b) Exonuclease III Figure 4.2 (P57) P54-86

7 (a) S1 nuclease The reactions catalysed by different types of endonuclease (b) Dnase I (c) A restriction endonuclease Figure 4.3 (P58) P54-86

8 (a) Discontinuity repair The two reactions catalysed by DNA ligase (b) Joining two molecules Figure 4.4 (P59) P54-86

9 (a) The basic reaction The reactions catalysed by DNA polymerase (b) DNA polymerase I (c) The Klenow fragment (d) Reverse transcriptase Figure 4.5 (P59) P54-86

10 (a) Alkaline phosphatase The reactions catalysed by DNA modifying enzymes (b) Polynucleotide kinase (A) Terminal deoxynucleotidyl transferase Figure 4.6 (P61) P54-86

11 Topoisomerases have yet find a real use in genetic engineering P54-86

12 Restriction endonucleases: Enzymes for cutting DNA (a) Vector molecules (b) The DNA molecule containing the gene to be cloned The need for very precise cutting manipulations in a gene cloning experiment Figure 4.7 (P62) P54-86

13 The discovery & function of restriction endonucleases (a) Restriction of phage DNA (b) Bacterial DNA is not cleaved The function of a restriction endonuclease in a bacterial cell Figure 4.8 (P63) P54-86

14 The recognition sequnces of restriction endonucleases P65 P54-86

15 Restriction endonucleases (a) Blunt ends (b) Sticky ends (c) Same sticky ends pproduced by different restriction endonucleases The ends produced by cleavage of DNA with diferent restriction enzyme Figure 4.9 (P66) P54-86

16 Restriction endonucleases (a) Cleavage sites on λ DNA (b) Fragment sizes Restriction of the λ molecule Figure 4.10 (P67) P54-86

17 Restriction endonucleases Performing a restriction digest in the laboratory Figure 4.11 (P68) P54-86

18 Electrophoresis (a) Standard electrophoresis (b) Gel electrophoresis Figure 4.12 (P71) P54-86

19 Visualizing DNA Visualizing DNA bands in agarose Figure 4.13 (P72) P54-86

20 Visualizing DNA Visualizing radioactively labelled DNA Figure 4.14 (P73) P54-86

21 Visualizing i DNA Radioactively labelling Figure 4.15 (P74) P54-86

22 The size of DNA Estimation of the size of DNA Figure 4.16 (P75) P54-86

23 Isolation of DNA fragments based on a restriction map Using a restriction map for digestion Figure 4.17 (P76) P54-86

24 Restriction map Figure 4.18 (P77) P54-86

25 Joining of DNA molecules together Ligation: the final step in construction of a recombinant DNA Figure 4.19 (P79) P54-86

26 Joining of DNA molecules together (a) Ligating blunt ends (b) Ligating sticky ends The different joining reactions catalysed by DNA ligase Figure 4.20 (P79) P54-86

27 Putting sticky ends onto a blunt-ended molecular (a) A typical linker (b) The use of linkers Linkers and their use Figure 4.21 (P80) P54-86

28 A possible problem of the use of linkers A possible problem of the use of linkers Figure 4.22 (P82) P54-86

29 Adaptors and the potential problem with the use Adaptors and the potential problem with the use Figure 4.23 (P82) P54-86

30 The 5 and 3 termini of a polynucleotide The distinction between the 5 and 3 termini of a polynucleotide Figure 4.24 (P83) P54-86

31 The use of adaptors The use of adaptors Figure 4.25 (P84) P54-86

32 Ch 1 What is gene cloning? The basic steps in gene cloning: 1. Vector (Ch2 (Ch.2, 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 1.1 (P5)

33 Ch 5 Introduction of DNA into Living Cells The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) Transformation 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Selection (Ch. 5) P87-106

34 Ch 5 Introduction of DNA into Living Cells The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Selection (Ch. 5) Transformation Figure 5.1 (P88) P87-106

35 Ch 5 Introduction of DNA into Living Cells The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Bacterial cells Bacteria Phages Non-bacterial cells Animal Plant P87-106

36 Ch 5 Introduction of DNA into Living Cells (a) The product of ligation The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) (b) All circular molecules will be cloned 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.2 (P89) P87-106

37 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: The binding and uptake of 1. DNA Vector by a (Ch.2, (Ch2 competent 3, 6, 7) bacterial cell 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.3 (P91) P87-106

38 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: Selecting cells that containing pbr322 plasmids by plating onto 1. Vector agar medium (Ch.2, (Ch2 3, 6, 7) containing ampicillin and/or 2. Digestion tetracycline (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.4 (P92) P87-106

39 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: Phenotypic expression 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.5 (P93) P87-106

40 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: Insertional inactivation (LucZ or antibiotic resistance) 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.6 (P94) P87-106

41 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: The cloning vector pbr Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.7 (P95) P87-106

42 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: Screening for pbr322 recombinants by insertional inactivation 1. Vector of (Ch.2, (Ch2 tetrcycline 3, 6, 7) resistance gene 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.8 (P96) P87-106

43 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: The cloning vector puc8 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.9 (P97) P87-106

44 Ch 5 Introduction of DNA into Living Cells Plasmids The basic steps in gene cloning: The rationale behind insertional inactivation of the lacz gene carried by puc8 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.10 (P98) P87-106

45 Ch 5 Introduction of DNA into Living Cells The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) Phage DNA 2. Digestion (Ch. 4) Transfection=transformation 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) In vito packaging of λ cloning vectors 5. Selection (Ch. 5) Figure 5.10 (P98) P87-106

46 Ch 5 Introduction of DNA into Living Cells Phage DNA The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) In vito packaging Figure 5.11 (P100) P87-106

47 Ch 5 Introduction of DNA into Living Cells Phage DNA The basic steps in gene cloning: Bacteriophage plaques 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.12 (P101) P87-106

48 Ch 5 Introduction of DNA into Living Cells Phage DNA The basic steps in gene cloning: Strategies for selection of recombinant phage 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.13 (P102) P87-106

49 Ch 5 Introduction of DNA into Living Cells The basic steps in gene cloning: 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) Nonbacterial cells 4. Transformation (Ch. 5) 5. Selection (Ch. 5) P87-106

50 Ch 5 Introduction of DNA into Living Cells Nonbacterial cells The basic steps in gene cloning: Strategies for inducing new DNA into animal and plant 1. cells Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.14 (P104) P87-106

51 Ch 5 Introduction of DNA into Living Cells Nonbacterial cells The basic steps in gene cloning: Two physical methods for introducing DNA into cells 1. Vector (Ch.2, (Ch2 3, 6, 7) 2. Digestion (Ch. 4) 3. Ligation (Ch. 4) 4. Transformation (Ch. 5) 5. Selection (Ch. 5) Figure 5.15 (P105) P87-106