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Biotechnology Lecture Notes Outline Biol 201

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Biotechnology Lecture Notes Outline Biol 201 K. Marr - Fall 2006 Overview of Recombinant DNA technologies Injection of DNA or a nucleus into a cell – PowerPoint PPT presentation

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Title: Biotechnology Lecture Notes Outline Biol 201


1
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

2
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

3
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
4
Use of a Retrovirus for Gene Therapy
  • Applications
  • Somatic Gene Therapy to treat
  • Gaucher Disease
  • SCIDs Bubble Boy
  • (Severe Combined Immune Difficiency)

5
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

6
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

7
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

8
Figure 20.2 An overview of how bacterial
plasmids are used to clone genes
9
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)

10
Using a restriction enzyme and DNA ligase to make
recombinant DNA Figure 20.3
11
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.

12
Figure 20.8 Gel electrophoresis of macromolecules
13
Figure 20.9 Using restriction fragment patterns
to distinguish DNA from different alleles
14
DNA fingerprints from a murder case
  • Whose blood is on the defendants clothing?

15
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

16
The polymerase chain reaction (PCR) Figure 20.7
17
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)

18
3. Overview of how bacterial plasmids are used
to clone genes
Figure 20.2
19
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

20
Use of Reverse Transcriptase to make
complementary DNA (cDNA) of a eukaryotic gene
21
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

22
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.

23
Figure 20.10 Restriction fragment analysis by
Southern blotting
24
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?

25
Using Plasmids to Create Recombinant DNA
26
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.

27
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?

28
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)

29
Figure 20.5 Using a DNA probe to identify a
cloned gene in a population of bacteria
30
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!!

31
Human Gene Therapy using...
  1. Retroviruses
  2. Adenoviruses
  3. Liposomes
  4. Naked DNA

32
Use of a Retrovirus for Gene Therapy
  • Applications
  • Somatic Gene Therapy to treat
  • Gaucher Disease
  • SCIDs Bubble Boy
  • (Severe Combined Immune Difficiency)

33
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

34
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

35
Figure 20.6 Genomic libraries
36
Figure 20.11 Chromosome walking
37
Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 1)
38
Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 2)
39
Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 3)
40
Figure 20.12 Sequencing of DNA by the Sanger
method (Layer 4)
41
Figure 20.13 Alternative strategies for
sequencing an entire genome
42
Table 20.1 Genome Sizes and Numbers of Genes
43
Figure 20.14a DNA microarray assay for gene
expression
44
Figure 20.14b DNA microarray assay for gene
expression
45
Figure 20.15 RFLP markers close to a gene
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