Biotechnology Lecture Notes Outline Biol 201

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Biotechnology Lecture Notes OutlineBiol 201 K.
Marr - Fall 2006

• Overview of Recombinant DNA technologies
• Injection of DNA or a nucleus into a cell
• Gene Therapy
• Pharm Animals
• Genetic Modification of Plants (e.g. GM foods)
• Use of Prokaryotes to produce Eukaryotic gene
  products
• Overview of various techniques
• Use of Restriction Enzymes DNA Ligase to make
  recombinant DNA molecules
• Use of Gel Electrophoresis...
• To separate restriction fragments
• For DNA fingerprinting
• PCR (Polymerase Chain Reaction)

• Strategies used to Genetically Engineer Bacteria
• How to isolate specific genes using..
• RNA Probes
• Reverse Transcriptase
• Human Gene Therapy using...
• Retroviruses
• Adenoviruses
• Liposomes
• Naked DNA

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1. Overview of Recombinant DNA technologies

• Injection of DNA or a nucleus into a cell
• Gene Therapy
• Pharm Animals
• Genetic Modification of Plants (e.g. GM foods)
• Use of Prokaryotes to produce Eukaryotic gene
  products

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Injection of DNA or a nucleus into Cell

• Potential Applications
• Germ line Gene Therapyinject therapeutic gene
  into an egg cell (affects future generations)
• Somatic Gene TherapyInject therapeutic gene into
  a somatic cell, culture reinsert into an
  individual
• Cloninginject nucleus into an enucleated egg,
  culture implant into a surrogate mother.

Drawback Inefficient means of gene transfer
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Use of a Retrovirus for Gene Therapy

• Applications
• Somatic Gene Therapy to treat
• Gaucher Disease
• SCIDs Bubble Boy
• (Severe Combined Immune Difficiency)

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Transgenic Pharm animals

• Potential Applications
• Genetically modify mammals to produce therapeutic
  peptide drugs (e.g. insulin, )
• Isolate and purify drug from the milk
• Potentially a more cost effective method to
  produce pharmaceuticals

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Using the Ti plasmid as a vector for genetic
engineering in plants

• Potential Applications
• Genetically modify plants to...
• produce vaccines in their fruit (e.g. polio
  vaccine)
• be resistant to disease and pests
• require less fertilizer, pesticides and
  herbicides
• have a higher nutritional value

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Golden rice contrasted with ordinary rice

• Transgenic Rice
• Genetically modify plants to produce
  beta-carotene
• Beta Carotene is converted to vitamin A in humans
• Vitamin A deficiency leads to poor vision and
  high susceptibility to disease
• 70 of children lt5 years old in SE Asia suffer
  from vit. A deficiency

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Figure 20.2 An overview of how bacterial
plasmids are used to clone genes
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2. Overview of various techniques

• Use of Restriction Enzymes DNA Ligase to make
  recombinant DNA molecules
• Use of Gel Electrophoresis...
• To separate restriction fragments
• For DNA fingerprinting
• PCR (Polymerase Chain Reaction)

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Using a restriction enzyme and DNA ligase to make
recombinant DNA Figure 20.3
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Gel Electrophoresis

• A method of separating mixtures of large
  molecules (such as DNA fragments or proteins) on
  the basis of molecular size and charge.
• How its done
• An electric current is passed through a gel
  containing the mixture
• Molecules travel through the medium at a
  different rates according to size and electrical
  charge
• Rate a size and charge
• Agarose and polyacrylamide gels are the media
  commonly used for electrophoresis of proteins and
  nucleic acids.

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Figure 20.8 Gel electrophoresis of macromolecules
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Figure 20.9 Using restriction fragment patterns
to distinguish DNA from different alleles
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DNA fingerprints from a murder case

• Whose blood is on the defendants clothing?

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PCRPolymerase Chain Reaction

• A very quick, easy, automated method used to make
  copies of a specific segment of DNA
• Whats needed.
• DNA primers that bracket the desired sequence
  to be cloned
• Heat-resistant DNA polymerase
• DNA nucleotides
• Thermocycler

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The polymerase chain reaction (PCR) Figure 20.7
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3. Strategies used to Genetically Engineer
BacteriaSee fig. 20.2. An overview of how
bacterial plasmids are used to clone genes

• Isolate the gene of interest (e.g. insulin gene)
• Insert the gene of interest into a bacterial
  R-plasmid
• R-plasmids are circular DNA molecules found in
  some bacteria that provide resistance to up to 10
  different antibiotics
• Place the transgenic plasmid into bacterial cells
• Plasmid DNA reproduces each time the bacteria
  reproduce
• Culture the bacteria and isolate the gene product
  (e.g. insulin)

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3. Overview of how bacterial plasmids are used
to clone genes
Figure 20.2
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Step 1. How to Isolate the Gene of Interest

• Use Reverse Transcriptase to make the gene of
  Interest
• Method 1 (see figure on next slide)
• Isolate mRNA for the gene product of interest
  (e.g. Insulin mRNA)
• Use Reverse Transcriptase to produce cDNA
  (complementary DNA)
• Use PCR to clone the cDNA
• Separate the synthetic gene of interest by
  electrophoresis

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Use of Reverse Transcriptase to make
complementary DNA (cDNA) of a eukaryotic gene
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Step 1. How to Isolate the Gene of Interest

• Use Reverse Transcriptase to make the gene of
  Interest
• Method 2
• Determine the primary structure (i.e. the amino
  acid sequence) of the protein of interest (e.g.
  insulin) with an automated protein sequencer
• Use table of codons to determine the mRNA
  sequence
• Synthesize the mRNA in the lab
• Use Reverse Transcriptase to produce cDNA and PCR
  to clone the cDNA (as before)
• Separate the synthetic gene of interest by
  electrophoresis

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1. How to Isolate the Gene of Interest

• Use a labeled DNA Probe to Isolate Gene of
  Interest (Southern Blot Method? see next slide)
• Extract and purify DNA from cells
• Cut DNA with restriction enzyme (e.g. Eco R1)
• ? Whats a restriction enzyme? (fig.
  20.3)
• ? Note Must cut outside of gene w/o
  too much excess baggage
• Separate DNA fragments by gel electrophoresis
• Transfer DNA from the fragile gel to a nylon
  sheet and heat to sep. strands (fig. 20.10)
• Hybridize gene of interest with a radio-labeled
  DNA or mRNA probe and expose w/ film to locate
  gene
• ? How do these probes work? (fig. 20.10)
• Use PCR to clone the isolated gene of interest.

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Figure 20.10 Restriction fragment analysis by
Southern blotting
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Steps 2 3. How to Insert the Gene of Interest
into the R-Plasmid

• See next 3 figures and animation
• Lyse bacteria with detergent to release the
  R-plasmid (e.g. ampicillin resistance plasmid)
• Cut the plasmid with the same restriction enzyme
  used to isolate the gene of interest
• Mix plasmid with gene of interest and join the
  two with DNA ligase
• ? How does this work?
• Add the recombinant plasmid to a bacterial
  culture
• ? Induce bacteria to take up
  plasmid (transformation)
• Grow bacteria on agar plate containing an
  antibiotic (e.g. ampicillin)
• Isolate those bacterial colonies that contain the
  recombinant plasmid ? How?
• ? Only some of the bacteria take up a
  plasmidHow do you know which ones did?
• ? Not all plasmids are recombinant plasmidsHow
  do you find those that are?
• ? Only some of plasmids contain the gene of
  interestHow do you identify these?

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Using Plasmids to Create Recombinant DNA
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Using Plasmids to Create Recombinant DNA

• Digest a plasmid vector with a restriction enzyme
  (e.g. EcoRI) at a single site to produce two
  sticky ends.
• Digest human DNA with EcoRI to produce pieces
  with the same sticky ends
• Use Human DNA or cDNA copied from mRNA using
  reverse transcriptase from retroviruses.
• Mix the two samples and allow to hybridize.
• Some plasmids will hybridize with pieces of human
  DNA at the EcoRI site.
• Use DNA ligase is used to covalently link the
  fragments.

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Insertion of Recombinant Plasmids into
Prokaryotic Cells

1. Only some of the bacteria take up a plasmidHow
   do you know which ones did?
2. Not all plasmids are recombinant plasmidsHow do
   you find those that are?
3. Only some of plasmids contain the gene of
   interestHow do you identify these?

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Identification of cells containing plasmids

• Cells containing plasmids contain the ampicillin
  resistance gene
• Grow cells on medium containing ampicillin
• How do you know which colonies contain the gene
  of interest?
• Use a DNA probe (see fig. 20.5)

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Figure 20.5 Using a DNA probe to identify a
cloned gene in a population of bacteria
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Step 4. Culture Bacteria and Isolate Gene
Product

• Grow the recombinant bacteria in nutrient broth
  and isolate/purify the gene product from the
  broth
• Expensive to do, therefore mammals (e.g. cows and
  goats) are now being genetically modified to
  produce desired gene products in their milk!!

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Human Gene Therapy using...

1. Retroviruses
2. Adenoviruses
3. Liposomes
4. Naked DNA

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Use of a Retrovirus for Gene Therapy

• Applications
• Somatic Gene Therapy to treat
• Gaucher Disease
• SCIDs Bubble Boy
• (Severe Combined Immune Difficiency)

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Basic Strategies of Human Gene Therapy (1 of 2)

• Isolate and then clone the normal allele by PCR
• Insert normal allele into a disabled virus
• Retroviruses and adenoviruses are the most common
  vectors
• Retroviruses are much more efficient at forming a
  provirus, but have a greater chance of mutating
  to cause disease
• Adenoviruses are safer, but are relatively
  inefficient as a vector
• Liposomes (lipid spheres) are also used as
  vectors
• e.g. Gene therapy for Cystic Fibrosis involves
  using an inhaler to bring liposomes containing
  the CFTR gene to the cells lining the lungs)
• Infect host cells with recombinant virus

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Basic Strategies of Human Gene Therapy (2 of 2)

• Infect host cells with recombinant virus
• Add recombinant virus directly to individual
• e.g. Jesse Gelsinger
• Had Ornithine Transcarbamylase Deficiency Causes
  build up of ammonia in liver cells since they
  cannot convert the ammonia (toxic) produced by
  amino acid metabolism to urea (less toxic)
• Died in Sept.99 due to a severe immune response
  to the genetically modified adenovirus containing
  the OTC gene
• Isolate host cells from body and then add
  recombinant virus (e.g. blood stem cells in
  gene therapy for Gaucher disease)
• Inject genetically engineered cells back into the
  body

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Figure 20.6 Genomic libraries
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Figure 20.11 Chromosome walking
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Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 1)
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Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 2)
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Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 3)
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Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 4)
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Figure 20.13 Alternative strategies for
sequencing an entire genome
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Table 20.1 Genome Sizes and Numbers of Genes
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Figure 20.14a DNA microarray assay for gene
expression
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Figure 20.14b DNA microarray assay for gene
expression
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Figure 20.15 RFLP markers close to a gene