DNA TECHNOLOGY

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DNA TECHNOLOGY

• Genetic engineering and all that stuff

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Glowing mice
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Goals of Recombinant DNA Technology

• Eliminate undesirable phenotypic traits in
  humans, animals, plants, and microbes
• Combine beneficial traits of two or more
  organisms to create valuable new organisms
• Create organisms that synthesize products humans
  need

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Overview of recombinant DNA technology
Figure 8.1.1
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Overview of recombinant DNA technology
Figure 8.1.2
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Example plasmid (pBR322)
Plasmid Vectors
                                               
                                    
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Tools and Techniques

• Restriction Enzymes
• Cut DNA at precise locations based upon a
  specific target sequence of nucleotides
• DNA Ligase
• Enzyme that seals or ligates DNA pieces
  together

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Restriction Endonucleases

• Eco RI
• --GAATTC-- --G AATTC--
• --CTTAAG-- --CTTAA G--
• SmaI
• --CCCGGG-- --CCC GGG--
• --GGGCCC-- --GGG CCC--
• Recognition sequence Results of cutting

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  Using a restriction enzyme and DNA ligase to
make recombinant DNA
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Cloning Steps

• Isolate gene of interest (cut out of genome with
  a Restriction Enzyme)
• Cut Vector with same R.E. to get compatible ends
• Mix 2 DNAs together and Ligate (DNA Ligase)
• Transform
• Screen for successful event
• Celebrate

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  Cloning a human gene in a bacterial plasmid a
closer look
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Inserting DNA into Cells

• Goal of DNA technology is insertion of DNA into
  cell (transformation)
• Can use vectors and natural methods of
  transformation, transduction, and conjugation
• Artificial methods
• Electroporation
• Protoplast fusion
• Injection gene gun and microinjection

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Inserting DNA into Cells
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Inserting DNA into Cells
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Tools and Techniques

• Gel Electrophoresis

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Gel Electrophoresis
Figure 8.7a
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Gel Electrophoresis of Macromolecules
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Forensic use of DNA patternsDNA Fingerprints
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 Using restriction fragment patterns to
distinguish DNA from different alleles
RFLPs restriction fragment length polymorphisms
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 DNA fingerprints from a murder case
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DNA Fingerprinting
M mother F Father C children both
children show some bands with each parent
Figure 8.13
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Tools and Techniques

• Reverse transcriptase

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  Making complementary DNA (cDNA) for a
eukaryotic gene
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Tools and Techniques

• DNA synthesis
• DNA sequencing

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DNA sequencers
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Tools and Techniques

• PCR (Polymerase Chain Reaction)
• Large number of identical molecules of DNA are
  produced in vitro
• Allows any piece of DNA to be rapidly amplified !
• Only minute amounts of DNA are needed !

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PCR procedure

• Heat to 95 degrees to denature the DNA strands
• Cool and elongate with heat tolerant DNA
  Polymerase (e.g. Taq polymerase from Thermus
  aquaticus)
• Repeat cycle Repeat cycle Repeat cycle
  Repeat cycle Repeat cycle

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Animation Polymerase Chain Reaction
PLAY
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Applications

• Diagnosis of Disease
• Gene Therapy
• Vaccines and other Pharmaceuticals
• Forensic use
• Environmental use (bioremediation)
• Agricultural use
• Transgenic animals
• Transgenic plants

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Transgenic organisms
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 One type of Gene Therapy
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PHARM animals
Transgenic Sheep Carrying a human gene for a
human protein which they secrete in their milk.
The protein is then purified from their milk
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Golden rice contrasted with ordinary rice
Transgenic Golden Rice (High levels of
Beta-carotene used to make Vitamin A give the
rice a golden color.
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Using the Ti plasmid as a vector for genetic
engineering in plants
TRANSGENIC PLANT
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Safety and Ethics

• GMO (genetically modified organism) food
  controversy

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Scare tactics?
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Consumer distrustJustifiedsillysomewhere in
between?
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BT Corn basics

• BT protein is produced by the bacterium Bacillus
  thuringiensis. Toxic to some insects, nontoxic to
  all other life forms
• BT corn has the gene inserted so that the corn
  plant makes the BT protein.
• Ingestion of BT protein by the larvae of the
  European Corn Borer kills the larvae
• The corn plant now has its own defense

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Pluses and minuses

• Reduced need for pesticides
• Greater Yield, etc. etc.
• Potential impact on other species
• None of any consequence noted.but still the
  GREAT UNKNOWN.

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The Biotech Floodgate
TODAYs Facts (2008) 90 of all soybeans 75 of
all corn 65 of all foods on grocery store
shelves (soy and corn syrup are in all sorts of
stuff!)
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Whats Next

• Bananas that produce human vaccines
• Fish that mature more quickly
• Plants that produce plastics
• Fruit/nut trees that yield years earlier
• Crops that grow where they could not before
• Human milk from cows

Two featherless chickens peck around in some
grass at the Hebrew University in Rehovot.
Israeli scientists at the Agriculture department
of the university have genetically engineered
bare-skinned chickens as part of a research
project to develop succulent, low fat poultry
that is environmentally friendly. (AP)