Gene Cloning

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Gene Cloning
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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

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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.
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Gene cloning

• When DNA is extracted from an organism, all its
  genes are obtained
• In gene (DNA) cloning a particular gene is copied
  (cloned)

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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.

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Sources of DNA for Cloning

• 1) Chromosomal DNA
• 2) RNA converted to cDNA
• 4) PCR-amplified DNA

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PCR-amplified DNA
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Cloning Tools

• Restriction endonucleases
• Ligase
• Vectors
• Host
• Methods for introducing DNA into a host cell

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Cutting DNA

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

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Blunt Sticky ends
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Pasting DNA

• Complementary ends (sticky ends) H-bond
• Ligase forms phosphodiester bond to seal strands
  together.

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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
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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)

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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.

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Plasmid vectors
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Bacteriophage vectors

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

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l vectors

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

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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)

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l?ZAP
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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

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BAC vector

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

Figure 5.5
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YAC vector

• Capable of carrying inserts of 200 - 2000 kbp in
  yeast

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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?

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Expression vector
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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

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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

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Blue/White Color Screening
lacZ
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Selecting Colonies with Recombinant Plasmids
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Colony hybridization

• DNA probe available?
• part of same gene
• orthologue from another species
• synthetic oligonucleotide

Figure 6.12
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End
Sayonara