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Gene Cloning

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A method for identifying and purifying a particular DNA fragment (clone) of ... gene-source DNA into the cloning vector using the same restriction enzyme; bind ... – PowerPoint PPT presentation

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Title: Gene Cloning


1
Gene Cloning
2
Cloning - a definition
  • From the Greek - klon, a twig
  • An aggregate of the asexually produced progeny of
    an individuala group of replicas of all or part
    of a macromolecule (such as DNA or an antibody)
  • An individual grown from a single somatic cell of
    its parent genetically identical to it
  • Clone a collection of molecules or cells, all
    identical to an original molecule or cell

3
DNA CLONING
A method for identifying and purifying a
particular DNA fragment (clone) of interest from
a complex mixture of DNA fragments, and then
producing large numbers of the fragment (clone)
of interest.
4
Gene cloning
  • When DNA is extracted from an organism, all its
    genes are obtained
  • In gene (DNA) cloning a particular gene is copied
    (cloned)

5
Why Clone DNA?
  • A particular gene can be isolated and its
    nucleotide sequence determined
  • Control sequences of DNA can be identified
    analyzed
  • Protein/enzyme/RNA function can be investigated
  • Mutations can be identified, e.g. gene defects
    related to specific diseases
  • Organisms can be engineered for specific
    purposes, e.g. insulin production, insect
    resistance, etc.

6
Sources of DNA for Cloning
  • 1) Chromosomal DNA
  • 2) RNA converted to cDNA
  • 4) PCR-amplified DNA

7
PCR-amplified DNA
8
Cloning Tools
  • Restriction endonucleases
  • Ligase
  • Vectors
  • Host
  • Methods for introducing DNA into a host cell

9
Cutting DNA
  • Restriction endonucleases (restriction enzymes)
  • sticky ends
  • blunt ends
  • Nomenclature
  • EcoRI
  • E genus (Escherichia)
  • co species (coli)
  • R strain
  • I of enzyme

10
Blunt Sticky ends
11
Pasting DNA
  • Complementary ends (sticky ends) H-bond
  • Ligase forms phosphodiester bond to seal strands
    together.

12
Cloning vectors
allowing the exogenous DNA to be inserted,
stored, and manipulated mainly at DNA level.
1 Plasmid vectors 2 Bacteriophage vectors 3
Cosmids 4 BACs YACs
13
Plasmid vectors
Plasmid vectors are double-stranded, circular,
self-replicating, extra-chromosomal DNA molecules.
  • Advantages
  • Small, easy to handle
  • Straightforward selection strategies
  • Useful for cloning small DNA fragments
  • (lt 10kbp)
  • Disadvantages
  • Less useful for cloning large DNA fragments
  • (gt 10kbp)

14
A plasmid vector for cloning
  • Contains an origin of replication, allowing for
    replication independent of hosts genome.
  • Contains Selective markers Selection of cells
    containing a plasmid
  • twin antibiotic resistance
  • blue-white screening
  • Contains a multiple cloning site (MCS)
  • Easy to be isolated from the host cell.

15
Plasmid vectors
16
Bacteriophage vectors
  • Advantages
  • Useful for cloning large DNA fragments
  • (10 - 23 kbp)
  • Inherent size selection for large inserts
  • Disadvantages
  • Less easy to handle

17
l vectors
  • Left arm
  • head tail proteins
  • Right arm
  • DNA synthesis
  • regulation
  • host lysis
  • Deleted central region
  • integration excision
  • regulation

18
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19
Cosmid vectors
combine the properties of plasmid vectors with
the useful properties of the l cos site
  • Advantages
  • Useful for cloning very large DNA fragments
  • (32 - 47 kbp)
  • Inherent size selection for large inserts
  • Handle like plasmids
  • Disadvantages
  • Not easy to handle very large plasmids ( 50 kbp)

20
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21
l?ZAP
22
BACs and YACs
BACs Bacterial Artificial Chromosomes YACs
Yeast Artificial Chromosomes
  • Advantages
  • Useful for cloning extremely large DNA fragments
  • (100 - 2,000 kbp)
  • This is very important for genome sequencing
    projects
  • Disadvantages
  • Not easy to handle extremely large DNA molecules

23
BAC vector
  • oriS and oriE mediate replication
  • parA and parB maintain single copy number
  • ChloramphenicolR marker

Figure 5.5
24
YAC vector
  • Capable of carrying inserts of 200 - 2000 kbp in
    yeast

25
What determines the choice vector?
  • insert size
  • vector size
  • restriction sites
  • copy number
  • cloning efficiency
  • ability to screen for inserts
  • what down-stream experiments do you plan?

26
Expression vector
27
How to clone DNA
  • Isolation of cloning vector (bacterial plasmid)
    gene-source DNA (gene of interest)
  • Insertion of gene-source DNA into the cloning
    vector using the same restriction enzyme bind
    the fragmented DNA with DNA ligase
  • Introduction of cloning vector into cells
    (transformation by bacterial cells)
  • Cloning of cells (and foreign genes)
  • Identification of cell clones carrying the gene
    of interest

28
Screening of the clone
  • The medium in this petri dish contains the
    antibiotic Kanamycin
  • The bacteria on the right contain Kanr, a plasmid
    that is resistant to Kanamycin, while the one on
    the left has no resistance
  • Note the difference in growth

29
Blue/White Color Screening
lacZ
30
Selecting Colonies with Recombinant Plasmids
31
Colony hybridization
  • DNA probe available?
  • part of same gene
  • orthologue from another species
  • synthetic oligonucleotide

Figure 6.12
32
End
Sayonara
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