Recombinant DNA and Genetic Engineering

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?