Recombinant DNA

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Recombinant DNA
AulanniamBiochemistry LaboratoryChemistry
LaboratoryBrawijaya University
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Definition

• To put DNA from one organism into the DNA of
  bacteria

Methods
Transformation Transduction Conjugation
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Method Transformation

• The modification of the genotype of a cell
  (usually prokaryotic) by introducing DNA from
  another source
• The uptake of DNA from an organisms environment
• The uptake and expression of DNA in a bacterium

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Method Transformation

• 3 elements required
• Suitable host
• Self-replicating vector
• Means of selecting of host cell that have taken
  up the gene
• Plasmids
• Small, circular, extra-chromosomal DNA molecules
  found in bacteria which can replicate on their
  own outside of a host cell
• Rapid growth rate
• Cloning limit 0.1-10 kb

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Method Transformation

• Plasmid (cont.)
• Fertility-(F-)plasmids, which contain only
  tra-genes. Their only function is to initiate
  conjugation.
• Resistance-(R-)plasmids, which contain genes that
  can build a resistance against antibiotics or
  poisons.
• Col-plasmids, which contain genes that code for
  (determine the production of) colicines, proteins
  that can kill other bacteria.
• Degrative plasmids, which enable the digestion of
  unusual substances, e.g., toluole or salicylic
  acid.
• Virulence plasmids, which turn the bacterium into
  a pathogen.

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Method Transformation

• The modification of the genotype of a cell
  (usually prokaryotic) by introducing DNA from
  another source
• The uptake of DNA from an organisms environment
• Cosmids
• Extrachromosomal circular DNA molecules that
  combines features of plasmids and phage
• Cloning limit 35-50 kb

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Method Transduction

• Process of transfection with phage
  introduction(equivalent to transformation)
• Phages
• Derivatives of bacteriophage lambda
• Virus which infects E. coli
• Linear molecules
• Transformation efficiency 100X greater than
  plasmid vector
• Cloning limit 8-25 kb

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Method Conjugation

• Bacterial Artificial Chromosomes (BAC)
• Based on bacterial mini-F plasmids
• Small pieces of episomal bacterial DNA that give
  the bacteria the ability to initiation
  conjugation with adjacent bacteria
• Cloning limit 75-300 kb

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Definitions for rDNA process

• Restriction endonuclease
• Natural function
• to protect bacteria by cutting and inactivating
  the DNA of infecting viruses
• rDNA
• Recognize specific base sequences and cleave the
  DNA molecule
• EcoR1 cuts at GAATTC
• BamHI cuts CCTAGG
• HindIII cuts TTCGAA

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Definitions for rDNA process

• Sticky ends
• Single stranded extensions of DNA created from a
  restriction enzyme
• Cloning Vector
• A DNA molecule that carries foreign DNA into a
  host cell, where it replicated
• Phages
• Plasmid
• Cosmid

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Definitions for rDNA process

• Donor cells
• Cell supplying the snippet of DNA
• Recipient cells
• The cell supplying the cell as well as the rest
  of the bacterial chromosome
• DNA ligase
• Enzyme used to bind sticky ends

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rDNA Steps (generalized)

1. Plasmid isolated and cut with enzyme
2. DNA isolated from donor cell and cut with same
   enzyme
3. Donor and plasmid DNA mixed and sealed with DNA
   ligase
4. rDNA introduced into a bacterium
5. Replication of plasmids

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Results

• 3 results
• Cells that lack plasmids (picked up human DNA)
• Cells that contain plasmid that do not contain a
  foreign gene (picked up plasmid DNA)
• Cells that contain plasmid that have picked up a
  foreign gene (picked up rDNA)

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Distinguishing rDNA

• Antibiotic resistance marker(s) added to plasmid
  (ex. Ampicillin and tetracycline genes)
• Human DNA only sensitive to both
• Plasmid DNA only resistant to both
• rDNA resistant to ampicillin but sensitive to
  tetracycline (the human DNA is inserted into the
  tetracycline coding sequence, thus disrupting the
  gene)

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Other info

• Gene isolation
• Genomic library
• collection of recombinant bacteria that harbor
  pieces of a genome
• DNA probe (gene sequence in question)
• A labeled short sequence of DNA that corresponds
  to a specific gene.
• Emits a signal when it binds to its complement in
  a bacterial cell that contains a recombinant
  plasmid

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Applications

• Epidermal growth factor
• Accelerates healing of wounds and burns
• treats gastric ulcers
• Insulin
• Allows cells to take up glucose in treatment of
  type I diabetes mellitus
• Renin inhibitor
• Lowers blood pressure
• Interleukin-2
• Treats kidney cancer