1_ch 20 dna technology_campbell 6e_f06

8/8/2019 1_Ch 20 DNA Technology_Campbell 6e_F06

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Biotechnology Lecture Notes OutlineBiol 201 K. Marr - Fall 2006

1. Overview of Recombinant DNA

technologiesa. Injection of DNA or a nucleus into a cell

b. Gene Therapy

c. Pharm Animals

d. Genetic Modification of Plants (e.g. GM

foods)

e. Use of Prokaryotes to produceEukaryotic gene products

2. Overview of various techniques

a. Use of Restriction Enzymes & DNA

Ligase to make recombinant DNAmolecules

b. Use of Gel Electrophoresis...

To separate restriction fragments

For DNA fingerprinting

c. PCR (Polymerase Chain Reaction)

3. Strategies used to

Genetically EngineerBacteria

How to isolate specific genes using..

RNA Probes

Reverse Transcriptase

4. Human Gene Therapyusing...

a. Retroviruses

b. Adenoviruses

c. Liposomes

d. Naked DNA


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1. Overview of Recombinant DNA technologies

a. Injection of DNA or a nucleus into a cell

b. Gene Therapy

c. Pharm Animals

d. Genetic Modification of Plants (e.g. GM foods)e. Use of Prokaryotes to produce Eukaryotic gene products


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Injection of DNA or a nucleus into Cell

Potential Applications

1. Germ line Gene Therapyinject therapeutic gene into an egg cell(affects future generations)

2. Somatic Gene TherapyInject therapeutic gene into a somatic cell, culture & reinsert into an

individual

3. 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 ApplicationsGenetically 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


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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

a. Use of Restriction Enzymes & DNA Ligase to make

recombinant DNA molecules

b. Use of Gel Electrophoresis...

To separate restriction fragments

For DNA fingerprinting

c. 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

1. A method of separating mixtures of large molecules

(such as DNA fragments or proteins) on the basis ofmolecular size and charge.

2. 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 E size and charge

Agarose and polyacrylamide gels are the media commonlyused 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.

1. DNA primers that bracket the desired sequence to be

cloned

2. Heat-resistant DNA polymerase

3. DNA nucleotides

4. Thermocycler


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The polymerase chain

reaction (PCR)

Figure 20.7


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3. Strategies used to Genetically Engineer Bacteria

See fig. 20.2. An overview of how bacterial plasmids are used to clone genes

1. Isolate the gene of interest (e.g. insulin gene)

2. 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 differentantibiotics

3. Place the transgenic plasmid into bacterial cells

Plasmid DNA reproduces each time the bacteria reproduce

4. 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 tomake the gene ofInterest

Method #1 (see figure on next slide)

1. Isolate mRNA for the gene product of interest (e.g. Insulin

mRNA)

2. Use Reverse Transcriptase to produce cDNA (complementary

DNA)

3. Use PCR to clone the cDNA

3. 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 tomake the gene ofInterest

Method #2

1. Determine the primary structure (i.e. the amino acid sequence)of the protein of interest (e.g. insulin) with an automated proteinsequencer

2. Use table of codons to determine the mRNA sequence

3. Synthesize the mRNA in the lab

4. Use Reverse Transcriptase to produce cDNA and PCR toclone the cDNA (as before)

5. 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 ofInterest(Southern Blot Method see next slide)

1. Extract and purify DNA from cells

2. 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

3. Separate DNA fragments by gel electrophoresis

4. Transfer DNA from the fragile gel to a nylon sheet and heat to sep. strands (fig. 20.10)

5. Hybridize gene of interest with a radio-labeled DNA* ormRNA* probe and expose w/film to locate gene

How do these probes work? (fig. 20.10)

6. 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

3. Mix plasmid with gene of interest and join the two with DNA ligase

How does this work?

4. Add the recombinant plasmid to a bacterial culture

Induce bacteria to take up plasmid (transformation)

5. Grow bacteria on agar plate containing an antibiotic (e.g. ampicillin)

6. 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

1. Digest a plasmid vector with a restriction enzyme (e.g.EcoRI) at a single site to produce two sticky ends.

2. Digest human DNA with EcoRI to produce pieces with thesame sticky ends

Use Human DNA or cDNA copied from mRNA using reversetranscriptase from retroviruses.

3. Mix the two samples and allow to hybridize.

Some plasmids will hybridize with pieces of human DNA at theEcoRI site.

4. 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 takeup a plasmidHow do youknow which ones did?

2. Not all plasmids arerecombinant plasmidsHowdo you find those that are?

3. Only some of plasmids containthe 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...

a. Retroviruses

b. Adenoviruses

c. Liposomes

d. 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)

1. Isolate and then clone the normal allele by PCR

2. 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 agreater 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 bringliposomes containing the CFTR gene to the cells lining the lungs)

3. Infect host cells with recombinant virus


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3. Infect host cells with recombinant virus

a. 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

b. 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

Basic Strategies of Human Gene Therapy (2 of 2)


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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 20.13 Alternative strategies for sequencing an entire genome

T bl 20 1 G Si d N b f G


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Table 20.1 Genome Sizes and Numbers of Genes

Fi 20 14 DNA i f i


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Figure 20.14a DNA microarray assay for gene expression

Fi 20 14b DNA i f i


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Figure 20.14b DNA microarray assay for gene expression

Fi 20 15 RFLP k l t


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Figure 20.15 RFLP markers close to a gene

1_ch 20 dna technology_campbell 6e_f06

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