Download Chapter 14 Genetic Engineering PP Notes

Chapter 14: Genetic Engineering
-Modification of the DNA of an organism to
produce new genes with new
characteristics
Biotechnology
• Use of organisms to benefit humanity
Recombinant DNA technology
• DNA from different organisms is spliced
together
• Allows scientists to make many copies of any
DNA segment (clone)
• Can introduce foreign DNA into cells of
microorganisms
Recombinant DNA technology
• Restriction enzymes – cut DNA
– Bacteria produce for defense against viruses
• Vector – transports DNA into a cell
– Ex: bacteriophage
– Plasmid – separate, smaller circular DNA that
maybe be present and able to replicate inside
bacteria
• Transformation – uptake of foreign DNA by
cells
– How plasmids can get into bacteria
Bacterial
Conjugation
and
Recombination
Recombinant DNA technology
• Palindromic sequences – reads the same as
complement, in opposite direction
– AAGCTT
– TTCGAA
– Many restriction enzymes cut these sequences
• Restriction enzymes cut on a stagger  sticky
ends (can pair with complementary singlestranded end of other DNA cut with same
enzyme)
• DNA Ligase – links 2 fragments 
recombinant DNA
Fig. 20-3-3
Restriction site
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Restriction Enzymes
Steps of Creating a Recombinant
DNA Plasmid (Basic)
• 1. Plasmids and desired DNA cut by same
restriction enzyme
• 2. Mix 2 types of DNA so sticky ends pair
• 3. DNA ligase forms bonds between fragments
Fig. 20-2
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
DNA of
chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
Interest
Protein expressed
by gene of interest
Copies of gene
Basic
Protein harvested
4 Basic research and
various applications
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Cloning a Gene
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying recombinant
plasmid with disrupted lacZ gene
One of many
bacterial
clones
Cloning DNA
• Genome – total DNA per cell
• Genomic library – collection of DNA fragments
more or less representative of all DNA in
genome
• Genetic Probe – single stranded DNA or RNA
that is radioactively labeled and can attach to
target sequence by base pairing rules
• A probe can be synthesized that is complementary
to the gene of interest
• For example, if the desired gene is
5 … G G C T A A C T T A G C … 3
– Then we would synthesize this probe
3 C C G A T T G A A T C G 5
DNA Probe
Using a DNA
probe
Polymerase Chain Reaction (PCR)
• Can amplify a small sample of DNA quickly
• DNA replication in vitro
• 2 strands separated by heating so special
heat-resistant DNA polymerase called Taq
polymerase used (thermophile)
• MAJOR BONUS: Only specific sequences can
be replicated
• Study: crime scenes, archaeological remains
PCR
Gel Electrophoresis
• Separates fragments like DNA, RNA or
polypeptides (they carry charge and can
migrate in an electrical field
• RNA and DNA (-) --- so they move to (+) pole
• Smaller fragments go further
• Compare sample to standard
• Usually “blot” - transfer DNA from gel to
nitrocellulose filter for further analysis
• DNA Fingerprinting
Gel Electrophoresis
Fig. 20-9a
TECHNIQUE
Power
source
Mixture of
DNA molecules of
different
sizes
Anode
– Cathode
+
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
Fig. 20-9b
RESULTS
DNA Fingerprint
Transgenic Organisms
• Plants and animals in which foreign genes
have been incorporated
• Animals
– Inject DNA into nucleus of egg or stem cell
– Eggs implanted in uterus; stem cells injected into
blastocysts + then implanted into foster mother
• Plants
– Disease resistance
– Pesticide resistance
Transgenics
Fig. 20-20
Embryonic stem cells
Early human embryo
at blastocyst stage
(mammalian equivalent of blastula)
Adult stem cells
From bone marrow
in this example
Cells generating
all embryonic
cell types
Cells generating
some cell types
Cultured
stem cells
Different
culture
conditions
Different
types of
differentiated
cells
Liver cells
Nerve cells
Blood cells
Fig. 20-18
TECHNIQUE
Mammary
cell donor
Egg cell
donor
2
1
Egg cell
from ovary
Cultured
mammary cells
3 Cells fused
3
4 Grown in
Nucleus
removed
Nucleus from
mammary cell
culture
Early embryo
5 Implanted
in uterus
of a third
sheep
Surrogate
mother
6 Embryonic
development
RESULTS
Lamb (“Dolly”)
genetically identical to
mammary cell donor
Fig. 20-19
Cloning Video
GE Plants
Application of GE
• Human proteins
– Insulin
– Hormones - HGH
• Human treatments for disease
– Multiple sclerosis, certain cancers, heart attacks,
forms of anemia
• Vaccines
Fig. 20-23
Fig. 20-24
(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b) These and other STR data exonerated Washington and
led Tinsley to plead guilty to the murder.
Forensics