Download Recombinant DNA

Chapter 26
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1
26.1 DNA Cloning
• Knowledge of DNA has led to an ability to
manipulate the genes of organisms.
• Cloned genes are used to alter the genome of
viruses or cells.
– Bacterial, plant, or animal cells
• This practice, genetic engineering, has many
uses, from producing a product to treating
cancer and genetic disorders.
2
The Cloning of a Gene
• Cloning
– Production of identical copies of an organism
through asexual means
• Ex: bacteria from the same colony, identical twins
• Gene cloning
– Production of many identical copies of a single
gene
3
26.1 DNA Cloning
• Uses of gene cloning
– Produce large quantities of the gene’s protein
product
– Learn how a cloned gene codes for a particular
protein
– Alter the phenotypes of other organisms in a
beneficial way
• Produce transgenic organism
• Gene therapy - cloned genes are used to modify a
human
4
Recombinant DNA Technology
• Recombinant DNA (rDNA)
– Contains DNA from two or more different sources
– Vector – piece of DNA that foreign DNA can be
added to
• Plasmids are accessory rings of DNA in bacteria,
commonly used as vectors.
• They are not part of the bacterial chromosomes.
5
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA
duplex
A
G
A
A
T
T
C
G
C
T
C
T
T
A
A
G
C
G
6
Recombinant DNA Technology
• 2 enzymes needed to introduce foreign DNA
– Restriction enzyme – to cleave vector DNA
– DNA ligase – to seal two pieces of DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA A
duplex
T
G
A
A
T
T
C
G
C
C
T
T
A
A
G C
G
restriction
enzyme
A
A
G
T
C
A
"sticky ends"
T
T
A
T
T
C
G
C
G
C
G
A
7
Recombinant DNA Technology
• Restriction enzymes
– Hundreds occur naturally, found in bacteria
– Act to restrict the growth of viruses, as a primitive
immune system in bacteria
– Used in cloning as molecular scissors that cut DNA
at precise sequences
• Ex: EcoR1 always recognizes and cuts DNA at the
sequence GAATTC
8
Recombinant DNA Technology
• After cutting at the EcoR1 site, a gap is created in
which pieces of foreign DNA can be placed if there
is complementary pairing.
• DNA ligase will seal the foreign DNA into the vector.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA A
duplex
T
G
A
A
T
T
C
G
C
C
T
T
A
A
G C
G
restriction
enzyme
A
A
G
T
C
A
"sticky ends"
T
T
A
T
T
C
G
C
G
C
G
A
9
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
Figure 26.1
10
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
insulin gene
Figure 26.1
1. Restriction enzyme
cleaves DNA.
11
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
insulin gene
1. Restriction enzyme
cleaves DNA.
2. DNA ligase seals
human gene and
plasmid.
recombinant DNA
Figure 26.1
12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
insulin gene
1. Restriction enzyme
cleaves DNA.
2. DNA ligase seals
human gene and
plasmid.
recombinant DNA
3. Host cell takes up
recombined plasmid.
Figure 26.1
13
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human DNA
plasmid
bacterium
human cell
insulin gene
1. Restriction enzyme
cleaves DNA.
2. DNA ligase seals
human gene and
plasmid.
recombinant DNA
3. Host cell takes up
recombined plasmid.
4. Gene cloning occurs.
Figure 26.1
insulin
14
Please note that due to differing
operating systems, some animations
will not appear until the presentation is
viewed in Presentation Mode (Slide
Show view). You may see blank slides
in the “Normal” or “Slide Sorter” views.
All animations will appear after viewing
in Presentation Mode and playing each
animation. Most animations will require
the latest version of the Flash Player,
which is available at
http://get.adobe.com/flashplayer.
15
Polymerase Chain Reaction
• The polymerase chain reaction (PCR) can create
billions of copies of a segment of DNA in a test
tube in hours.
– Amplifies only specifically-targeted DNA sequence
– Targeted sequence is usually a few hundred bases
in length
– Uses DNA polymerase and DNA nucleotides
– Three basic steps that occur repeatedly usually for
about 35 to 40 cycles
16
Polymerase Chain Reaction
• Three basic steps in PCR occur repeatedly.
1. Denaturation – heated at 95°C for separation
2. Annealing – usually between 50 and 60°C to
allow the binding of a primer on the end of each
DNA strand
3. Extension – occurs at 72°C where a unique DNA
polymerase adds complementary bases to each
of the single DNA strands, creating a doublestranded DNA
17
Polymerase Chain Reaction
• PCR is a chain reaction because the targeted
DNA is repeatedly replicated.
• The amount of DNA doubles with each cycle.
• Automation is possible because of the use of a
temperature-insensitive DNA polymerase
extracted from Thermus aquaticus, a bacteria that
lives in hot springs.
• The enzyme tolerates the high temperature used
to separate the DNA strands (950C).
18
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
Figure 26.2
19
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
second
2
Figure 26.2
new
old
20
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
second
2
third
4
Figure 26.2
new
new
old
old
21
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
second
2
third
4
fourth
8
Figure 26.2
new
new
old
old
old
new
22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PCR
cycles
DNA
copies
first
1
second
2
third
4
fourth
8
fifth
16
new
new
old
old
old
new
and so forth
Figure 26.2
23
DNA Analysis
• DNA analysis has improved over time.
– Previous method involved entire genome being
treated with restriction enzymes
– Fragments separated by gel electrophoresis
• Smaller fragments move faster than larger ones
– Result was distinctive pattern of bands, called
DNA fingerprint
24
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Mother
fewer
repeats
Child
Male 1
Male 2
Fig. 26.3
more
repeats
Figure 26.3a
DNA Band patterns
25
DNA Analysis
• Short tandem repeat (STR) profiling used now
– STRs are the same short sequence of DNA bases that
recur several times.
• GATAGATAGATA
– Fragments are different lengths because each person
has their own number of repeats at the particular
location of the STR on the chromosome.
– The more STR loci employed, the more confident
scientists can be of distinctive results for each person.
26
DNA Analysis
• STR profiling
–
–
–
–
PCR is used to amplify target sequences of DNA
Creates fluorescently-labeled PCR products
Runs through automated DNA sequencer
Laser detects and records lengths of DNA
fragments
– Homozygotes have a single fragment
– Heterozygotes will have two fragments of different
lengths
27
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Mother
Child
Male 1
Male 2
fewer
repeats
more
repeats
a.
Fluorescence units
DNA Band patterns
Increasing size
Figure 26.3
b. Automated DNA finger printing
28
Biotechnology Products
• Transgenic bacteria, plants, and animals are
often called genetically modified organisms
(GMOs)
– Products they produce are biotechnology products.
29
Transgenic Bacteria
• Transgenic bacteria are produced by
recombinant DNA technology.
–
–
–
–
Grown in large vats called bioreactors
Bacteria express the cloned gene
Gene product collected from the media
Products include insulin, human growth hormone,
tPA, and hepatitis B vaccine
30
Transgenic Bacteria
• Other uses of transgenic bacteria
– Bacteria have been altered from frost-plus to
frost-minus bacteria.
• As a result, new crops such as frost-resistant
strawberries are being developed.
– Bacteria that colonize the roots of corn plants
have been engineered to have genes whose
products are toxic to insects that may damage
the roots.
31
Transgenic Bacteria
• Transgenic bacteria
– Can be selected for
their ability to
degrade a particular
substance
– Ability can be
enhanced by
bioengineering
– Eat oil, remove sulfur
from coal
Figure 26.5
32
Transgenic Plants
• Foreign genes can be introduced.
– Immature plant embryos
– Protoplasts – plant cells with cell wall removed
• Exposed to an electric current while in a liquid
containing foreign DNA
• Self-sealing pores are formed that allow the desired
DNA to enter
• Go on to develop into mature plants that express
the foreign gene
33
Transgenic Plants
• The pomato is one result of this technology.
– Produces potatoes below ground and tomatoes
above ground
• Pest resistance in cotton, corn, and potato
strains can be created.
• Soybeans are resistant to herbicide.
• Plants can also be engineered to produce
human proteins.
34
Transgenic Animals
• Technology has been developed to insert genes
into eggs of animals.
– It is possible to microinject foreign genes into eggs by
hand or by vortex mixing.
– Eggs are placed in an agitator with DNA and siliconcarbide needles.
– The needles make tiny holes in the eggs allowing the
DNA to enter.
– When the eggs are fertilized, transgenic offspring are
produced.
• The gene for bovine growth hormone (BGH) has been inserted
to produce larger fishes, cows, pigs, rabbits, and sheep
35
Transgenic Animals
• Gene pharming
– Transgenic farm animals can be used to produce
pharmaceuticals.
– Proteins can be harvested from animals’ milk.
– Plans exist to produce drugs for treatment of
cystic fibrosis, cancer, blood diseases.
36
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human gene
for growth
hormone
microinjection of human gene
donor of egg
development within
a host goat
human growth
hormone
Transgenic goat produces
human growth hormone.
milk
Figure 26.6a
37
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
transgenic goat cells
with gene for human
growth hormone
microinjection of
these 2n nuclei into
enucleated donor eggs
enucleated eggs
donor of eggs
development
within
host goats
milk
Figure 26.6b
Cloned
transgenic
goats produce
human growth
hormone.
38
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
human gene
for growth
hormone
microinjection of human gene
donor of egg
development within
a host goat
human growth
hormone
Transgenic goat produces
human growth hormone.
milk
a.
transgenic goat cells
with gene for human
growth hormone
microinjection of
these 2n nuclei into
enucleated donor eggs
enucleated eggs
donor of eggs
development
within
host goats
milk
Figure 26.6
b.
Cloned
transgenic
goats produce
human growth
hormone.
39
26.3 Gene Therapy
• Gene therapy is the insertion of genetic material
into human cells for the treatment of genetic
disorders, cardiovascular disease and cancer.
• Various methods of gene transfer have been used.
– Viruses, genetically modified to be safe, can be used to
introduce a normal gene into the body.
– Liposomes, microscopic globules of lipids, can also be
used to introduce normal genes.
– Sometimes the gene is injected directly into a specific
region of the body.
40
Ex vivo Gene Therapy
• Ex vivo method for treating SCID (severe
combined immunodeficiency)
– Used for children who lack the enzyme ADA (adenosine
deaminase), involved in the maturation of T and B cells
– They are prone to constant infections; may die without
treatment
• Gene therapy treatment steps
– Remove bone marrow stem cells from body
– Infect cells with a virus that carries the normal gene that
codes for the enzyme, ADA
– Return cells to patient with the hope they will divide
expressing the normal gene for ADA
41
Ex vivo Gene Therapy
• Treatment of familial hypercholesterolemia
– Liver cells lack a receptor protein for removing
cholesterol from the blood.
– High blood cholesterol levels make a patient
subject to heart attacks at young age.
– A liver portion is surgically excised and then
infected with a virus containing a normal gene for
the receptor.
– The liver portion is returned to patient.
42
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Brain
(gene transfer by injection)*
• Huntington disease
• Alzheimer disease
• Parkinson disease
• brain tumors
Skin
(gene transfer by modified
blood cells)**
• skin cancer
Lungs
(gene transfer by aerosol spray)*
• cystic fibrosis
• hereditary emphysema
Liver
(gene transfer by modified
implants)**
• familial hypercholesterolemia
Blood
(gene transfer by bone
marrow transplant)*
• sickle-cell disease
Endothelium
(blood vessel lining)
(gene transfer by
implantation of
modified implants)**
• hemophilia
• diabetes mellitus
Muscle
(gene transfer by injection)*
• Duchenne muscular dystrophy
Bone marrow
(gene transfer by implantation
of modified stem cells)**
• SCID
• sickle-cell disease
Figure 26.7
* invivo
** ex vivo
43
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1. Remove bone
marrow stem cells.
defective gene
Figure 26.8
44
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
2. Use retroviruses
to bring the normal
gene into the bone
marrow stem cells.
1. Remove bone
marrow stem cells.
retrovirus
defective gene
viral
recombinant
RNA
normal gene
Figure 26.8
45
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
2. Use retroviruses
to bring the normal
gene into the bone
marrow stem cells.
1. Remove bone
marrow stem cells.
retrovirus
defective gene
4. Return genetically
engineered cells
to patient.
viral recombinant DNA
reverse transcription
viral recombinant RNA
viral
recombinant
RNA
normal gene
3. Viral recombinant
DNA carries normal
gene into genome.
normal gene
Figure 26.8
46
In Vivo Gene Therapy
• In vivo therapy
– Cystic fibrosis patients lack a gene coding for chloride
transporter protein.
– A thick mucus forms in the lungs, leading to infections
of the respiratory tract.
• Treatment
– The gene needed to cure cystic fibrosis is sprayed
into the nose or delivered to the lower respiratory tract
by an adenovirus vector or by using liposomes.
• Limited success so far
47
In Vivo Gene Therapy
• Increasingly relied upon as a part of
cancer treatment
– Used to make healthy cells more tolerant of
chemotherapy
– Making tumor cells more sensitive to
chemotherapy
48
26.4 Genomics, Proteomics, and
Bioinformatics
– Genetics in the 21st century largely concerns
genomics.
• Study of the complete genetic sequences of humans
and other organisms
– First step is knowing the sequence of bases in
genomes
» Of the 3.2 billion bases in the human genome, 99% is
noncoding and contains many repetitive sequences.
– Second step is mapping the location of genes on the
chromosomes
– Approximately 20,000 genes code for proteins
49
Sequencing the Genome
• Human Genome Project (HGP)
– The HGP was a13-year effort that involved both
university and private laboratories.
– We now know the sequence of the roughly 3.2
billion pairs of DNA bases in our genome.
– New DNA sequencing technology helped speed the
process.
– New genomes are being sequenced all the time and
at a much faster rate now.
• Recently, the African clawed frog was sequenced in less
than one year.
50
Sequencing the Genome
• Discovery of single nucleotide polymorphisms
(SNPs)
–
–
–
–
Difference of only one nucleotide between individuals
Many have no effect
Others contribute to protein-coding difference
May change susceptibility to disease or response to
medical treatments
– Raise the possibility of “designer drugs” tailored to
individual’s genotype
51
Sequencing the Genome
• The HGP, along with identification of RNAs in
cells, led to the determination that humans have
20,000-25,000 genes.
– Structural genomics - knowing the sequence of the
bases and how many genes we have
– Functional genomics – what does it code for?
• Most genes are expected to code for proteins.
• Noncoding or “junk DNA” may have important
functions.
52
Genome Architecture
• Genome architecture
– Nearly 99% of the human genome is DNA that
does not directly code for amino acid sequences.
– Some is transcribed into rRNA or tRNA.
• Both are involved in protein assembly.
– The rest of the genome consists of a variety of
sequences.
• Some are repeated, other not.
53
Genome Architecture
• Balance of the genome
– Transposable elements (or transposons)
• 45% of human genome
• Discovered by Barbara McClintock in 1950
• Thought to be driving force in evolution
– Repetitive DNA elements
• Same sequence of two or more nucleotides
repeated
• May not be useless – telomeres have this structure
– Sequences with unknown function
54
Redefining the Gene
• What is a gene?
– Historically, a gene was thought of as a
particular location (locus) of a chromosome.
– Eukaryotic genes appear to be randomly
distributed along chromosomes.
– Genes are fragmented into exons.
• 95% or more of most human genes are introns.
• Introns may be regulators of gene expression.
– HGP has changed the way researchers define the
concept of a “gene”.
55
Redefining the Gene
• What is a gene?
– Modern definition focuses on result of transcription.
– A gene is a genomic sequence (either DNA or
RNA) directly encoding functional products, either
RNA or protein.
• Gene product may not necessarily be a protein
• Gene may not be found at a particular locus on a
chromosome
• Genetic material need not be only DNA—some
prokaryotes have RNA genes
56
Functional and Comparative
Genomics
• Comparative genomics
– Compare genomes of organisms
– Identify similarities between the sequence of human
bases and those of other organisms
– Provide way to study genome changes through time
• Track evolution of HIV
– Understand the evolutionary relationships among
organisms
• Human and chimpanzee 98% alike
• Human and mouse 85% alike
57
58
Functional and Comparative
Genomics
• Functional genomics
– Understand the function of the various genes
discovered within each genomic sequence and
how these genes interact
– Help deduce the function of human genes by
comparison to other genomes
– Use a microarray to tell what genes are turned on
in a specific cell or tissue type in a particular
organism at a particular point in time and under
certain environmental circumstances
59
Proteomics
– Proteomics is the study of the structure, function,
and interactions of cellular proteins.
• Proteins differ depending on each cell type.
• Each cell produces hundreds of different proteins
that can vary between or within cells depending on
conditions.
– Computer modeling of the three-dimensional shape
of these proteins is important.
• Protein shape and function is essential to the
discovery of better drugs.
60
Bioinformatics
• Bioinformatics
– Computer technologies, specially developed software,
and statistical techniques can be used to study
biological information, particularly databases that
contain much genomic and proteomic information.
– Bioinformatics can find significant patterns in the raw
data of DNA sequences.
– Computers can help make correlations between
genomic differences among large numbers of people
and certain diseases.
61
Bioinformatics
• BLAST - stands for basic local alignment
search tool
– BLAST is used to identify homologous genes
among the genomic sequences of model
organisms.
– Homologous genes code for the same proteins,
though the base sequence may be slightly different.
– Finding these differences can help identify the
putative function of genes as new organisms’
genomes are sequenced.
62