Download Cell with DNA containing gene of interest

Genetics of Cancer
Fig. 11-12
Signaling cell
Signal
Transduction:
Signaling
molecule
Plasma
Receptor membrane
protein
1
2
3
Way in which a cell
can respond to
signals from its
environment
Results in a change
in which genes are
expressed (turned on)
Target cell
Relay
proteins
Transcription
factor
(activated)
4
Nucleus
DNA
5
mRNA Transcription
New
protein
6
Translation
Fig. 11-20b
Growth-inhibiting
factor
Receptor
Relay
proteins
Transcription
factor
(activated)
Normal tumorsuppressor genes
prohibit cell division
Nonfunctional transcription
factor (product of faulty p53
tumor-suppressor gene)
cannot trigger
transcription
Normal product
of p53 gene
Transcription
Protein that
inhibits
cell division
Translation
Protein absent
(cell division
not inhibited)
Fig. 11-20a
Growth factor
Receptor
Target cell
Hyperactive
relay protein
(product of
ras oncogene)
issues signals
on its own
Normal product
of ras gene
Relay
proteins
Oncogenes
STIMULATE
cell
division
Transcription
factor
(activated)
DNA
Nucleus
Protein that
Stimulates
cell division
Transcription
Translation
Ras is an
oncogene
(cancer gene)
the normal form
of the gene is a
proto-oncogene
Fig. 11-18b
Tumor-suppressor gene
Mutated tumor-suppressor gene
Normal
growthinhibiting
protein
Defective,
nonfunctioning
protein
Cell division
under control
Cell division not
under control
Progression of Colon Cancer
6
A tissue comprised of billions of cells heterozygous for BRCA1 or BRCA2
Both alleles of
BRCA1 or both
alleles of
BRCA2 must
be mutant for
cancer to
develop.
Why would in
follow a dominant
inheritance
pattern?
Your (my) probability of winning the lottery is very small. The
probability that someone will win it is very large.
8
One of the key tools in DNA
technology is the restriction enzyme
Where do these restriction
enzymes come from????
What is their natural function???
How can we use them???
Recombinant DNA
• DNA from 2 sources combined
– Can be used to clone genes
– Used to produce a particular protein
E. coli bacterium
Plasmid
1
Bacterial
chromosome
Isolate
plasmid
Cell with DNA
containing gene
of interest
2
Isolate
DNA
DNA
Gene of interest
A plasmid is a small circular piece of DNA found in some bacterial cells
Separate from main chromosome
May have genes that give the bacteria an advantage in certain
circumstances
Bacteria can take up plasmids from their environment
E. coli bacterium
Plasmid
Bacterial
chromosome
1
Cell with DNA
containing gene
of interest
Isolate
plasmid
2
Isolate
DNA
DNA
3
Gene of interest
Cut plasmid
with enzyme
4
Cut cell’s DNA
with same
Gene
of interest
enzyme
E. coli bacterium
Plasmid
Bacterial
chromosome
1
Cell with DNA
containing gene
of interest
Isolate
plasmid
3
2
Cut plasmid
with enzyme
Isolate
DNA
DNA
Gene of interest
4
Cut cell’s DNA
with same enzyme
Gene
of interest
5 Combine targeted fragment
and plasmid DNA
E. coli bacterium
Plasmid
Bacterial
chromosome
1
Cell with DNA
containing gene
of interest
Isolate
plasmid
3
2
Cut plasmid
with enzyme
Isolate
DNA
DNA
Gene of interest
4
Cut cell’s DNA
with same enzyme
Gene
of interest
Recombinant
DNA
plasmid
5
Combine targeted fragment
and plasmid DNA
6
Add DNA ligase,
which closes
the circle with
covalent bonds
Gene
of interest
Recombinant
DNA
plasmid
Gene
of interest
7
Recombinant
bacterium
Put plasmid
into bacterium
Recombinant
DNA
plasmid
Gene
of interest
7
Recombinant
bacterium
7
Allow bacterium
8 to reproduce
Clone
of cells
-gene of interest
has also been cloned
Put plasmid
into bacterium
by transformation
Examples of
gene use
Genes may be inserted
into other organisms
Recombinant
DNA
plasmid
Gene
of interest
9
7
Put plasmid
into bacterium
by transformation
Recombinant
bacterium
8
Allow bacterium
to reproduce
Genes or proteins
are isolated from the
cloned bacterium
Harvested
proteins
may be
used directly
Clone
of cells
Examples of
protein use
Important to use the same restriction enzyme to
cut each source of DNA
This allows complementary sticky ends to be
created that can later base-pair to combine the DNA
Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
Two (or more)
fragments stick
together by
base-pairing
4
3
Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
4
DNA ligase
pastes the strands
5
Recombinant
DNA molecule
Steps in cloning a gene
1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that
cuts in one place, opening the circle
4. DNA with the target gene is treated with the same
enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate
with each other
Copyright © 2009 Pearson Education, Inc.
Steps in cloning a gene
6. Recombinant DNA molecules are produced when
DNA ligase joins plasmid and target segments
together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of
cells
Copyright © 2009 Pearson Education, Inc.
Problem: if we’re trying to
get a bacterium
(prokaryote) to make our
proteins, bacteria do not
have introns… so, they
can’t remove them
Solution: Use reverse
transcriptase (found in
retroviruses) to make
DNA from mature mRNA
Use restriction enzymes to break
DNA into manageable sized pieces
that we can separate
What can we tell from this?
• It can be used to compare the DNA from
different organisms
• Used to detect disease alleles
• Used to “match” DNA samples
– Determine parentage
– Crime scene forensics
Fig. 12-11
Crime scene
1 DNA isolated
2 DNA of selected
markers amplified
3 Amplified DNA
compared
Suspect 1
Suspect 2
PCR is used to amplify DNA sequences
• http://learn.genetics.utah.edu/content/labs/p
cr/
– Mix ingredients in a thermocycler
• What do you need to make lots of copies of DNA?
Copyright © 2009 Pearson Education, Inc.
Detecting disease alleles
Fig. 12-14a
STR site 1
STR site 2
Crime scene DNA
Number of short tandem Number of short tandem
repeats match
repeats do not match
Suspect’s DNA
Fig. 12-14b
Crime scene
DNA
Suspect’s
DNA
Cycle 1
yields 2 molecules
Genomic
DNA
3
1
3
5
3
Target
sequence
5
5
5
3
Cycle 2
yields 4 molecules
5
5
2 Cool to allow
3
Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
3
5
3
Primer
3
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 3
yields 8 molecules
Cycle 1
yields 2 molecules
Genomic
DNA
3
3
5
5
3
Target
sequence
5
3
3
5
5
3
2 Cool to allow
1 Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
5
3
5
3
Primer
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules