Recombinant DNA and Genetic Engineering

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14 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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Recombinant DNA
and Genetic
Engineering

Chapter 16

Familial Hypercholesterolemia


Gene encodes protein that serves as cell’s LDL
receptor


Two normal alleles for the gene keep blood level
of LDLs low


Two mutated alleles lead to abnormally high
cholesterol levels & heart disease

Example of Gene Therapy


Woman with familial hypercholesterolemia


Part of her liver was removed


Virus used to insert normal gene for LDL receptor
into cultured liver cells


Modified liver cells placed back in patient


Results of Gene Therapy


Modified cells alive in woman’s liver


Blood levels of LDLs down 20 percent


No evidence of atherosclerosis


Cholesterol levels remain high


Remains to be seen whether procedure will
prolong her life

Genetic Changes


Humans have been changing the genetics of
other species for thousands of years


Artificial selection of plants and animals


Natural processes also at work


Mutation, crossing over

Genetic Engineering


Genes are isolated, modified, and inserted into
an organism


Made possible by recombinant technology


Cut DNA up and recombine pieces


Amplify modified pieces

Discovery of Restriction Enzymes



Hamilton Smith was studying how
Haemophilus
influenzae

defend themselves from bacteriophage
attack



Discovered bacteria have an enzyme that chops
up viral DNA

Specificity of Cuts


Restriction enzymes cut DNA at a specific
sequence



Number of cuts made in DNA will depend
on number of times the “target” sequence
occurs

Making Recombinant DNA

5’

3’

G

C T T A A

A A T T C

G

G

A A T T C

C T T A A

G

3’

5’

one DNA fragment

another DNA fragment

3’

5’


In
-
text
figure

Page 254

Making Recombinant DNA

nick

5’

3’

3’

5’

G

A A T T C

C T T A A

G

nick

G

A A T T C

C T T A A

G

DNA ligase action


In
-
text
figure

Page 254

Using Plasmids


Plasmid is small circle of bacterial DNA


Foreign DNA can be inserted into plasmid



Forms recombinant plasmids


Plasmid is a cloning vector


Can deliver DNA into another cell

Using Plasmids

DNA

fragments

+

enzymes

recombinant

plasmids

host cells containing
recombinant plasmids

Figure 16.4

Page 255

Amplifying DNA


Fragments can be inserted into

fast
-
growing microorganisms



Polymerase chain reaction (PCR)

Polymerase Chain Reaction


Sequence to be copied is heated


Primers are added and bind to ends of single
strands


DNA polymerase uses free nucleotides to
create complementary strands


Doubles number of copies of DNA

Polymerase
Chain Reaction

Double
-
stranded

DNA to copy

DNA heated to

90
°


94
°
C

Primers added to

base
-
pair with

ends

Mixture cooled;

base
-
pairing of

primers and ends

of DNA strands

DNA polymerases

assemble new

DNA strands

Figure 16.6

Page 256

Stepped Art

Polymerase
Chain Reaction

Figure 16.6

Page 256

Stepped Art

Mixture heated again;
makes all DNA
fragments unwind

Mixture cooled; base
-
pairing between
primers and ends of
single DNA strands

DNA polymerase
action again
doubles number of
identical DNA
fragments


DNA Fingerprints


Unique array of DNA fragments


Inherited from parents in Mendelian fashion


Even full siblings can be distinguished from one
another by this technique

Tandem Repeats


Short regions of DNA that differ
substantially among people


Many sites in genome where tandem repeats
occur


Each person carries a unique combination of
repeat numbers

Gel Electrophoresis


DNA is placed at one end of a gel


A current is applied to the gel


DNA molecules are negatively charged and
move toward positive end of gel


Smaller molecules move faster than larger ones

Analyzing DNA Fingerprints


DNA is stained or made visible by use of a
radioactive probe


Pattern of bands is used to:


Identify or rule out criminal suspects


Identify bodies


Determine paternity

Genome Sequencing


1995
-

Sequence of bacterium
Haemophilus
influenzae

determined


Automated DNA sequencing now main method


Draft sequence of entire human genome
determined in this way

Gene Libraries


Bacteria that contain different
cloned DNA fragments


Genomic library


cDNA library

Engineered Proteins


Bacteria can be used to grow medically valuable
proteins


Insulin, interferon, blood
-
clotting factors


Vaccines


Cleaning Up the Environment


Microorganisms normally break down
organic wastes and cycle materials


Some can be engineered to break down
pollutants or to take up larger amounts of
harmful materials

The Ti plasmid


Researchers
replace tumor
-
causing genes with
beneficial genes


Plasmid transfers
these genes to
cultured plant cells

foreign gene

in plasmid

plant cell

Figure 16.11

Page 261

Engineered Plants


Cotton plants that display resistance to herbicide


Aspen plants that produce less lignin and more
cellulose


Tobacco plants that produce human proteins


Mustard plant cells that produce biodegradable
plastic

First Engineered Mammals


Experimenters used mice with hormone
deficiency that leads to dwarfism


Fertilized mouse eggs were injected with gene
for rat growth hormone


Gene was integrated into mouse DNA


Engineered mice were 1
-
1/2 times larger than
unmodified littermates

Cloning Dolly


1997
-

A sheep cloned from an adult cell


Nucleus from mammary gland cell was
inserted into enucleated egg


Embryo implanted into surrogate mother


Sheep is genetic replica of animal from
which mammary cell was taken

Designer Cattle


Genetically identical cattle embryos can be
grown in culture


Embryos can be genetically modified


create resistance to mad cow disease


engineer cattle to produce human serum
albumin for medical use

The Human Genome Initiative

Goal
-

Map the entire human genome


Initially thought by many to be a waste of
resources


Process accelerated when Craig Ventner used
bits of cDNAs as hooks to find genes


Sequencing was completed ahead of schedule in
early 2001

Genomics


Structural genomics: actual mapping and
sequencing of genomes of individuals


Comparative genomics: concerned with possible
evolutionary relationships of groups of
organisms

Using Human Genes


Even with gene in hand it is difficult to
manipulate it to advantage


Viruses usually used to insert genes into
cultured human cells but procedure has
problems


Very difficult to get modified genes to work
where they should

Can Genetically Engineered
Bacteria “Escape”?



Genetically engineered bacteria are designed
so that they cannot survive outside lab


Genes are included that will be turned on in
outside environment, triggering death

Ethical Issues


Who decides what should be “corrected”
through genetic engineering?


Should animals be modified to provide
organs for human transplants?


Should humans be cloned?