Download Control of Gene Expression

What are the Techniques of
Biotechnology ?
• Restriction Endonucleases: enzymes that cut DNA at
specific codes (nucleotide sequences)
– Can buy from suppliers: ex. cut at ATATAT
• DNA Fingerprinting: sequence code of sample DNA (a
portion of genome) or compare digested samples via
gel electrophoresis
– In crime and paternity testing, evidence sample compared to
suspects’ samples; if different codes, or different patterns on gel,
then cannot be “donor”; if match, % likelihood based on size of
genome sequenced, or frequency of gel pattern in population
• Rape charges even filed against “unknown person” with sample DNA (statute of
limitations was approaching)
• DNA very strong evidence for innocence, not as strong for guilt; but has been
used as primary evidence in capital cases (resulted in executions)
– DNA from whale-meat in Japanese restaurants showed many whale
and dolphin species sold despite moratorium on most species
• Polymerase chain reaction: machine that replicates a small
sample of DNA into a larger amount of identical sample
(enough to work with)
Fig. 20.3
1
Restriction site
DNA
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
Cell containing gene
of interest
Bacterium
Fig.
20.2
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
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
DNA in
nucleus
Fig. 20.6
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase Poly-A tail
DNA Primer
strand
Degraded
mRNA
DNA
polymerase
cDNA
Fig. 20.9a
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Power
source
– Cathode
Anode +
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
5
TECHNIQUE
3
Target
sequence
Fig. 20.8
3
Genomic DNA
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
What are the Applications of
Biotechnology?
• Human Genome Project: highly collaborative; completed in early 2001;
human genome mapped and sequenced; next step is understanding
the functions of genes (and resulting proteins)
– Reasoning was that several genetic diseases would become better understood during the project (sooner than if each was studied independently)
• Genetic Screening: geneticists use interviews and DNA fingerprinting;
concerns regarding insurance and potential discrimination
• Genetic Therapy: inject “healthy genes” into blood; some success in
diseases of the blood (immune disorders)
• Genetic Engineering (recombinant technology): manipulate genes in
fertilized egg; replace un-wanted gene with copy of desired gene
– Transgenic Organisms: because genetic code and ribosome “machinery”
shared in all organisms, bacteria (and other organisms) can make human
proteins if appropriate gene is inserted into cell (or fertilized egg); such
proteins are often medicines (ex., replace casein gene in milk of sheep or
goats with desired gene)
– Agricultural Applications: genes for natural insecticides, drought-resistance,
and frost-resistance transferred to crops
Chromosome
bands
Cytogenetic map
Fig. 21.2
1
Genes located
by FISH
Linkage mapping
Genetic
markers
2
Physical mapping
Overlapping
fragments
3
DNA sequencing
1 Cut the DNA
into overlapping
fragments short enough
for sequencing
2 Clone the fragments
in plasmid or phage
vectors.
3 Sequence each
fragment.
4 Order the
sequences into
one overall
sequence
with computer
software.
Fig. 21.3
Fig. 20.10
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
Cloned
gene
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4
Inject engineered
cells into patient.
Bone
marrow
Fig.
20.22
TECHNIQUE
Agrobacterium tumefaciens
Fig. 20.25
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
RESULTS
Recombinant
Ti plasmid
Plant with new trait