1. Section G Gene manipulation
Molecular Biology Course
Section G Gene manipulation

2. G1-5 Screening libraries G1-6 Analysis of a clone
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. G1 DNA cloning: an overview
Gene manipulation
G1 DNA cloning: an overview

4. G1-1 DNA cloning (definition)
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. General features of a Vector
G1 DNA cloning: An Overview
General features of a Vector
autonomously replicating DNA independent of host’s genome.
Easily to be isolated from the host cell
Most are circular, some are linear
Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not.
Contains a multiple cloning site (MCS)

7. Types of vectors Cloning vectors Expression vectors
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
Expression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required.
bacterial expression vectors
yeast expression vectors
mammalian expression vectors
MCS

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 libraries are 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 clone is a genetically distinct individual or set of identical individuals
Genomic libraries cDNA libraries

15. Genomic libraries cDNA libraries G1 DNA cloning: An Overview
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 transcription are 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. G1-5 Screening libraries
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. Identify the protein product of an interested gene
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.
Sequencing the 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. G2 Preparation of plasmid DNA
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. G2-2 Plasmid minipreparation from E. coli
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. Miniprep G2 Preparation of plasmid DNA
Growth of the cells containing plasmids
Collect the 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. G2-3 Alkaline lysis G2 Preparation of plasmid DNA
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
G2- 6 Cesium chloride gradient centrifugation
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. G3 Restriction Enzymes and electrophoresis
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. G3-1 Restriction endonuclease
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. G3-2&3 Restriction sequences&Cohesive ends
G3 Restriction enzymes and electrophoresis
G3-2&3 Restriction sequences&Cohesive ends
5’ protruding ends
3’ protruding ends
Cohensive ends
p -GGG-3’
OH-CCC-5’
SmaI
5’-CCCGGG-3’
3’-GGGCCC-5’
5’-CCC-OH
3’-GGG- p +
blunting ends

30. Recognition sequences
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. G3-4 Restriction digestion
G3 Restriction enzymes and electrophoresis
G3-4 Restriction
digestion

32. G3-5 Agrose gel electrophoresis
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. Agrose gel electrophoresis
G3 Restriction enzymes and electrophoresis
Agrose gel electrophoresis

34. Isolation of fragments and Agarose gel electrophoresis
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 Ligation, transformation and analysis of recombinants
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. G4-1 Alkaline phophatse Single restriction enzyme directed cloning
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. G4-2&3 DNA ligation & recombinant DNA molecules
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. Recombinant DNA molecules
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. G4-4&5 Transformation and selection
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 is adequate for a simple cloning.

42. G4-6 Transformation efficiency
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 is adequate for a simple cloning.

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

44. Distinguish the recombinant plasmids from the recreated vectors by
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