Section G Gene manipulation

1. Molecular Biology Course Section G Gene manipulation

2. Gene manipulation G1-1 DNA cloning G1-2 Hosts and vectors G1-3 Subcloning G1-4 DNA libraries G1-5 Screening libraries G1-6 Analysis of a clone

3. Gene manipulation G1 DNA cloning: an overview

4. G1 DNA cloning: An Overview G1-1 DNA cloning (definition) DNA cloning is to place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector, forming recombinant DNA, which can be replicates independently of the original genome, and normally in other host species altogether. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organism, or a clone. This process is called DNA cloning.

5. G1 DNA cloning: An Overview G1-2 Hosts and vectors • Host organism/cell:where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. • Hosts for DNA cloning vector • Prokaryotic host : E. coli ( most cases) • Eukaryotic host : Yeast Saccharomyces cerevisiae (large fragments of human genome)

6. G1 DNA cloning: An Overview • General features of a Vector • autonomously replicating DNAindependent of host’s genome. • Easily to be isolatedfrom the host cell • Most are circular, some are linear • Contains at least oneselective marker, which allows host cells containing the vector to be selected amongst those which do not. • Contains amultiple cloning site (MCS)

7. G1 DNA cloning: An Overview Types of vectors • Cloning vectors • Expression vectors • Integration vectors

8. Cloning vectors:allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid-bacteriophage l hybrids (cosmids). Yeast cloning vector: yeast artificial chromosomes (YACs)

9. G1 DNA cloning: An Overview MCS • Expression vectors:allowing the exogenous DNA to be inserted and expressed. Promoterand terminator for RNA transcription are required. • bacterial expression vectors • yeast expression vectors • mammalian expression vectors

10. G1 DNA cloning: An Overview • Integration vectors:allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is conducted by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells. • bacterial integration vectors (Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) • yeast integration vectors • Mammalian integration vector: virus based

11. G1 DNA cloning: An Overview • G1-3 Subcloning • Transfer of a fragment of cloned DNA from one vector to another. • Enables us to investigate a short region of a large cloned fragment in more detail. • To transfer a gene from one plasmid to a vector designed to express it in a particular species.

12. DNA Subcloning: a flow chart Preparation of plasmids containing a cloned DNA fragment (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Restriction endonuclease Ligation (join the insert and the vector) Transformation & selection of transformants (introduce the plasmids into host cells) Assay of the recombinants

13. G1 DNA cloning: An Overview • Agrose Gel Electrophoresis: • check your DNA at each step • Separation and Purification of DNA fragments of interests • Analysis of recombinant plasmids ladder Restriction analysis of a plasmid

14. G1 DNA cloning: An Overview G1-4 DNA libraries • DNA librariesare sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. • A cloneis a genetically distinct individual or set of identical individuals • Genomic librariescDNA libraries

15. G1 DNA cloning: An Overview Genomic libraries prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA cDNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcriptionare inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding functional RNA or noncoding sequence.

16. G1 DNA cloning: An Overview G1-5 Screening libraries Searching the genes of interest in a DNA library • Hybridization to identify the interested DNA or its RNA product • Radiolabeled probes which is complementary to a region of the interested gene • Probes: • An oligonucleotide derived from the sequence of a protein product of the gene • A DNA fragment/oligo from a related gene of another species • Blotting the DNA or RNA on a membrane • Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane

17. G1 DNA cloning: An Overview • Identify the protein product of an interested gene • Protein activity • Western blotting using a specific antibody

18. G1 DNA cloning: An Overview G1-6 Analysis of a clone • Restriction mapping:digestion of the with restriction enzymes. • Sequencingthe cloned DNA You may have to fully understand the function and application of all the enzymes listed in Table 1 before the final exam

19. Gene manipulation G2 Preparation of plasmid DNA G2-1 Plasmid as vectors G2-2 Plasmid minipreparation G2-3 Alkaline lysis G2-4 Phenol extraction G2-5 Ethanol precipitation G2-6 Cesium chloride gradient (purification)

20. G2 Preparation of plasmid DNA G2-1 Plasmid as vectors • Plasmids:small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells. • contain an origin of replication and replicate independently • Usually carry a few genes, one of which may confer resistance to antibacterial substance. • Example:ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin.

21. G2 Preparation of plasmid DNA G2-2 Plasmid minipreparation from E. coli • Plasmids • ~2-20 kb in length that much smaller than E. coli chromosomal DNA (4600 kb), and independently supercoiled • Resistant to shearing force and chemical denaturation, thus can be isolated from the chromosomal DNA easily such as alkaline lysis. • Minipreparation (miniprep) • Isolation of plasmid DNA from a few mililiters (ml) of bacterial culture.

22. G2 Preparation of plasmid DNA Miniprep • Growth of the cells containing plasmids • Collectthe cells by centrifugation • Alkaline lysis • resuspension alkaline lysis  neutralization • Phenol extraction to get rid of the protein contaminants • Ethanol precipitation to concentrate the nucleic acids remained.(Please noted that RNase A is very bad for the lab working with RNA)

23. G2 Preparation of plasmid DNA • G2-3 Alkaline lysis • Resuspend the cells in a buffer solution • Lysozyme to digest the cell wall (optional) • Cell lysis in lysis buffer containing SDS (disrupts cell membrane and denatures proteins) and NaOH (denatures DNA) • Neutralization buffer containing KOAc (pH 5): renaturation of plasmid DNA (supercoiled) and precipitation of denatured proteins and chromosomal DNA which can not be renatured because of its size and physical property of easily being sheared.

24. Grow the cell Harvest the cell by centrifugation Resuspend the cell pellet Alkaline lysis of the cell neutralization Phenol extraction Ethanol precipitation CsCl gradient purification

25. G2- 6 Cesium chloride gradient centrifugation G2 Preparation of plasmid DNA • CsCl gradient purification is the last step of large scale plasmid DNA purification • Laborious • Best for the production of very pure supercoiled plasmid DNA • The presence of ethidium bromide (EB) is important. • Binding of EB to DNA will unwind the DNA and reduce the DNA density • Supercoilded DNA bind less EB than linear DNA or nicked DNA, thus has a higher density Supercoiled DNA may be purified from protein,RNA chromosomal DNA and nicked plasmid DNA in one step!!

26. Gene manipulation G3 Restriction Enzymes and electrophoresis G3-1 Restriction endonuclease G3-2 Recognition sequences G3-3 Cohesive ends G3-4 Restriction digests G3-5 Agarose gel electrophoresis G3-6 Isolation of fragments

27. G3 Restriction enzymes and electrophoresis G3-1 Restriction endonuclease • Bacterial enzymes which cut DNA into defined and reproducible fragments • Identified in the 1960s • Key discovery which allowed the DNA cloning to become a reality

28. G3 Restriction enzymes and electrophoresis • One component of the bacterial restriction-modification system,a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell • Restriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA) • Mythylase:methylates C or A of the cellular DNA

29. G3 Restriction enzymes and electrophoresis p -GGG-3’ OH-CCC-5’ G3-2&3 Restriction sequences&Cohesive ends 5’ protruding ends 3’ protruding ends Cohensive ends SmaI 5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-OH 3’-GGG- p + blunting ends

30. G3 Restriction enzymes and electrophoresis Recognition sequences • Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 46=4096 bp. • Highly specific Restriction enzymes • Commercially available • Require Mg2+ for enzymatic activity

31. G3 Restriction enzymes and electrophoresis G3-4 Restriction digestion

32. G3 Restriction enzymes and electrophoresis G3-5 Agrose gel electrophoresis Agrose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%) Negatively charged DNA - ve electrode + ve electrode

33. G3 Restriction enzymes and electrophoresis Agrose gel electrophoresis

34. G3 Restriction enzymes and electrophoresis Isolation of fragments and Agarose gel electrophoresis • Restriction digestion • Agarose gel electrophoresis insert • 3. Gel excision and purification • Ligation with vector • transformation

35. G4 Ligation, transformation and analysis of recombinants Gene manipulation G4-1 Alkaline phophatse G4-2&3 DNA ligation & recombinant DNA molecules G4-4&5 Transformation & selection G4-6 Transformation efficiency G4-7 Screening transformants G4-8 Growth and storage of transformants G4-9 Gel analysis G4-10 Fragment orientation

36. G4 Ligation, transformation and analysis of recombinants • G4-1 Alkaline phophatse • removes the phosphate groups from th 5’-ends of the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligation • Single restriction enzyme directed cloning

37. G4 Ligation, transformation and analysis of recombinants G4-2&3 DNA ligation & recombinant DNA molecules DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.

38. G4 Ligation, transformation and analysis of recombinants Recombinant DNA molecules X if the vector is phosphoralated

39. G4 Ligation, transformation and analysis of recombinants The use of alkaline phosphate to prevent religation of vector molecules

40. G4 Ligation, transformation and analysis of recombinants G4-4&5 Transformation and selection Competent cells: E. coli cells treated with Ca2+ solution are susceptible to take up exogenous DNA. Enzymes involved in host cell defending, such as restriction-modification system are suppressed. Transformation: a process of uptake of exogenous DNA by competent cells. Heat-shock: After the DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending

41. G4 Ligation, transformation and analysis of recombinants Selection with antibiotic resistance (ampr) Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103 to more than 108. 105 is adequate for a simple cloning.

42. G4 Ligation, transformation and analysis of recombinants G4-6 Transformation efficiency Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103 to more than 108. 105 is adequate for a simple cloning.

43. G4 Ligation, transformation and analysis of recombinants G4-7 Growth and storage of transformants • Can be grow in liquid broth or solid plates • Maintain theselection pressureby the presence of the corresponding antibiotics. Plasmid stability and lose • Storethe transformant bacteria by freezing a portion in the presence of glycerol to protect from ice crystal formation

44. G4 Ligation, transformation and analysis of recombinants G4-8&9 Gel analysis and fragment orientation • Distinguish the recombinant plasmids from the recreated vectors by • Size of the plasmids (not work well if the plasmids are prepared with alkaline lysis) • Restriction digestion • 2. Determine the orientation of a inserted DNA fragment cloned by a single enzyme by restriction digestion that cuts asymmetrically within the insert sequence, and once at a specific site of the vector

45. G4 Ligation, transformation and analysis of recombinants

46. G4 Ligation, transformation and analysis of recombinants 3. Analysis of a clone by restriction mapping

47. Thanks