Recombinant DNA Technology Lecture Notes

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Biotechnology Lecture Notes Outline

Biol 201


K. Marr
-

Fall 2006

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

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)

3.
Strategies used to
Genetically Engineer
Bacteria

How to isolate specific genes using..


RNA Probes


Reverse Transcriptase

4.
Human Gene Therapy
using...

a.
Retroviruses

b.
Adenoviruses

c.
Liposomes

d.
Naked DNA

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



Injection of DNA or a nucleus into Cell

Potential Applications

1.
Germ line Gene Therapy

in
ject therapeutic gene into an
egg cell

(affects future generations)

2.
Somatic Gene Therapy

Inject therapeutic gene into a
somatic cell
, culture & reinsert into an
individual

3.
Cloning

inject nucleus into an enucleated egg, culture & implant into a surrogate mother.

Drawback:

Inefficient means of gene transfer


Use of a Retrovirus
for Gene Therapy

Applications

Somatic Gene Therapy to treat


Gaucher Disease


SCID’s “Bubble Boy”

(
S
evere
C
ombined
I
mmune
D
ifficiency)

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


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

“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 <5 years old in SE Asia suffer from vit. A deficiency

Figure 20.2

An overview of how bacterial plasmids are used to clone genes

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)




Using a restriction enzyme and DNA
ligase to make recombinant DNA



Figure 20.3


Gel Electrophoresis

1.
A method of separating mixtures of large molecules
(such as DNA fragments or proteins) on the basis of
molecular size and charge.

2.
How it’s 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

獩s攠慮搠捨d牧r


Agarose and polyacrylamide gels are the media commonly
used for electrophoresis of proteins and nucleic acids.

Figure 20.8 Gel electrophoresis of

macromolecules

Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

DNA fingerprints from a murder case


Whose blood is on the defendant’s clothing?

PCR

Polymerase Chain Reaction


A very quick, easy, automated method used to
make copies of a specific segment of DNA


What’s needed….

1.
DNA primers that “bracket” the desired sequence to be
cloned

2.
Heat
-
resistant DNA polymerase

3.
DNA nucleotides

4.
Thermocycler

The polymerase chain
reaction (PCR)


Figure 20.7


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 different
antibiotics

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)

3. Overview of how bacterial plasmids are used to clone genes

Figure 20.2

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)

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

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

Step 1.

How to Isolate the Gene of Interest

Use Reverse Transcriptase to make the gene of Interest

Method #2

1.
Determine the primary structure

(i.e. the amino acid sequence)
of the protein of interest (e.g. insulin) with an automated protein
sequencer

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 to
clone the cDNA (as before)

5.
Separate the synthetic gene of interest by electrophoresis

1. How to Isolate the Gene of Interest

Use a labeled DNA Probe to Isolate Gene of Interest
(Southern Blot Method


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1.
Extract and purify DNA from cells

2.
Cut DNA with
restriction enzyme

(e.g. Eco R1)




What’s 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*
or

mRNA*

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.


Figure 20.10

Restriction fragment analysis by Southern blotting

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




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6.
Isolate those bacterial colonies that contain the recombinant plasmid


H潷
?




Only some of the bacteria take up a plasmid

How do you know which ones did?



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How do you find those that are?



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How do you identify these?




Using Plasmids to Create Recombinant DNA




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 the
same sticky ends


Use Human DNA or cDNA copied from mRNA using reverse
transcriptase from retroviruses.

3.
Mix the two samples and allow to hybridize.



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

4.
Use DNA ligase is used to covalently link the fragments.


Insertion of Recombinant Plasmids into Prokaryotic Cells


1.
Only some of the bacteria take
up a plasmid

How do you
know which ones did?

2.
Not all plasmids are
recombinant plasmids

How
do you find those that are?

3.
Only some of plasmids contain
the gene of interest

How do
you identify these?

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
)

Figure 20.5


Using a DNA probe to
identify a cloned gene in a
population of bacteria

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!!

Human Gene Therapy using...

a.
Retroviruses

b.
Adenoviruses

c.
Liposomes

d.
Naked DNA

Use of a Retrovirus
for Gene Therapy

Applications

Somatic Gene Therapy to treat


Gaucher Disease


SCID’s “Bubble Boy”

(
S
evere
C
ombined
I
mmune
D
ifficiency)

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 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)

3.
Infect host cells with recombinant virus

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)

Figure 20.6 Genomic libraries

Figure 20.11 Chromosome walking

Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)

Figure 20.12 Sequencing of DNA by the Sanger method (Layer 2)

Figure 20.12 Sequencing of DNA by the Sanger method (Layer 3)

Figure 20.12 Sequencing of DNA by the Sanger method (Layer 4)

Figure 20.13 Alternative strategies for sequencing an entire genome

Table 20.1 Genome Sizes and Numbers of Genes

Figure 20.14a DNA microarray assay for gene expression

Figure 20.14b DNA microarray assay for gene expression

Figure 20.15 RFLP markers close to a gene