Manipulation of genetic material
Summary
Tools for genetic engineering
Host/vector systems and DNA libraries
Genetic engineering experiment
Identifying genes in organisms
Analyzing DNA
Biotechnology and you
Genetic Engineering
Involves:
Identifying genes and
Creating “new” genes or “
designer genes
” through
genetic
recombination
.
Molecular Biologists’ Tools
Restriction endonucleases
–
enzymes borrowed from
nature.
Cut DNA at specific sites =
restriction sites
.
Uses: Cut DNA into smaller pieces for analysis and/or to
construct
recombinant DNA
molecules (addition of a
piece of foreign DNA to native DNA).
Molecular Biologists’ Tools
Restriction endonucleases cont’d
2 types:
type I
produces blunt end cuts,
type II
produces
staggered cuts with “sticky ends”.
Restriction sites
have certain base sequences called
palindromes, that are recognized by specific
endonucleases.
DNA Ligase
–
also borrowed from nature joins the
ends of cut strands.
GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS
Recognition
site
5
5
3
3
Plasmid
5
5
3
3
Recognition
site
Restriction
endonuclease
(EcoR1)
1.
Plasmid DNA
contains a recognition
site for a restriction
endonuclease.
2.
Attach the same
recognition site to the
gene that will be
inserted into the
plasmid.
3.
A restriction endonuclease
makes staggered cuts at each
of the recognition sites,
creating “sticky ends.”
Sticky end
Plasmid
Recombinant
plasmid
4.
Sticky ends on
plasmid and on gene to
be inserted bind by
complementary base
pairing.
5.
Use DNA ligase to
catalyze a phosphodiester
bond at points marked by
green arrows, “sealing” the
inserted gene.
GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS
Host/Vector Systems
Use: produce large amounts of recombinant DNA
quickly, storage of DNA.
Most common
host
is E.coli bacteria but other bacteria
and yeasts are also used. All reproduce quickly.
Vectors
:
plasmids
and
phages
carry recombinant DNA
into host.
Host/Vector Systems
Vectors
-
Plasmids
Small circular pieces of DNA into which foreign DNA
(10kb) can be inserted.
“Engineered” plasmids usually contain a gene for
antibiotic resistance and one for a metabolic enzyme.
Plasmids move in and out of the host’s DNA (bacteria)
and are replicated as the host reproduces.
Host/Vector Systems
Vectors
-
Phages
Derived from virus DNA, can accept larger pieces of
DNA (40 kb).
After “infection” the DNA becomes part of the host’s
DNA and is replicated.
Other vectors are used to infect animal or yeast cells.
DNA Libraries
Collection of host cells with vectors, each containing
pieces of DNA.
2 types
Genomic
–
fragments of DNA from the entire genome.
cDNA
–
fragments of “expressed” genes produced from
mRNAs using
reverse transcriptase.
Genetic Engineering Experiment
One of the 1
st
attempts was to produce a “safe” growth
hormone to treat pituitary dwarfism.
Procedure, fig 19.3:
Isolate mRNA from pituitary cells and use reverse
transcriptase to produce a piece of cDNA
(c=complementary).
Attach restriction sites to both ends of cDNA.
CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE
mRNA
mRNA
Single
-
stranded
cDNA
Reverse
transcriptase
Double
-
stranded
cDNA
3.
Make the cDNA double
-
stranded.
2.
Use reverse transcriptase to
synthesize a cDNA from each
mRNA.
1.
Isolate mRNAs from cells in pituitary
gland.
Genetic Engineering Experiment
Procedure cont’d
Cut cDNAs and plasmids with a restriction
endonuclease and insert cDNAs into plasmids (DNA
ligase).
Insert plasmids into bacteria.
Screen bacteria containing plasmids (& cDNA) by
growing in the presence of antibiotic. Those that grow
have antibiotic resistance carried by the plasmid and
form the cDNA library.
CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE
Recombinant
plasmid
cDNA
library
5.
Introduce recombinant plasmids
into
E. coli
cells via treatment that
makes cells permeable to DNA. Each
cell contains one type of recombinant
plasmid and thus one cDNA. The
collection of cells is the cDNA library.
4.
Insert each double
-
stranded
cDNA into a different plasmid (see
Figure 19.2).
Genetic Engineering Experiment
Screening of the library
-
Finding clones with cDNA
insert
cDNA is inserted within gene sequence for a metabolic
enzyme.
If bacteria DO NOT have a functional metabolic enzyme
they DO have a cDNA insert.
Genetic Engineering Experiment
Screening cont’d
-
Finding the “clones” with growth
hormone cDNA:
Transfer clones to filter paper.
Prepare a DNA “probe” with growth hormone sequence and a
radioactive marker (nucleotide).
Treat filter paper with probe which will “stick” or
hybridize
to
growth hormone cDNA.
Lay photographic film over filter paper. Clones with
radioactive probe will cause spots to appear on film.
Labeled probe
USING A DNA PROBE TO FIND A TARGET SEQUENCE IN
A COLLECTION OF MANY DNA SEQUENCES
1.
Single
-
stranded
DNA probe has a
label that can be
visualized.
2.
Expose probe
to collection of
single
-
stranded
DNA sequences.
3.
Probe binds to
complementary
sequences in target
DNA
—
and only to
that DNA. Target
DNA is now labeled
and can be isolated.
Finding Specific Genes by Probing a
cDNA
Library
SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE
1.
Grow
E. coli
cells
containing plasmids
on many plates. Each
colony contains a different
cDNA.
2.
Lay a filter on
each plate, then
remove. Some cells
from each colony
stick to filters.
Finding Specific Genes by Probing a
cDNA
Library
Labeled probe
3.
Treat bacteria
with chemicals to
break open cells
and make DNAs
single stranded.
4.
Probe filters
with labeled DNA
(short sequence
inferred from
amino acid
sequence of
growth hormone).
SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE
Finding Specific Genes by Probing a
cDNA
Library
6.
On original plates,
find colony of
E. coli
cells that contains
growth hormone
gene. Sample cells,
grow, and analyze.
5.
The labeled
probe DNA binds
to its
complementary
sequence in the
cDNA library.
E. coli
containing
growth hormone
gene
SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE
Identifying Genes in Organisms
Southern blotting
fig 19.8:
Isolate DNA from a new source and cut using restriction
endonucleases. Separate fragments using gel electrophoresis.
Transfer fragments to filter paper.
Treat filter paper with a DNA probe for a particular gene and
lay photographic film over filter paper.
If the gene is present, the probe will hybridize to a band and
will cause a black spot on film.
Location of restriction
endonuclease cuts
Sample 1
Samples from
four individuals
Double
-
stranded
DNA
Double
-
stranded
DNA
SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF
DNA FRAGMENTS
1.
Restriction endonucleases cut
DNA sample into fragments of
various lengths. Each type of
restriction endonuclease cuts a
specific sequence of DNA.
2.
A sample consists of
all the DNA fragments of
various lengths. The
sample is loaded into a
gel for electrophoresis.
3.
During electrophoresis,
a voltage gel separates
DNA fragments by size.
Small fragments run faster.
Power
supply
1
2
3
4
4.
The DNA fragments are treated to make
them single stranded.
3.
During electrophoresis, a voltage gel
separates DNA fragments by size. Small
fragments run faster.
Double
-
stranded
DNA
Single
-
stranded
DNA
Samples from
four individuals
1
2
3
4
1
2
3
4
Power
supply
SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET
DNA IN A LARGE COLLECTION OF DNA FRAGMENTS
Stack of
blotting paper
5.
Blotting. An alkaline
solution wicks up through
the gel into blotting
paper. DNA fragments
from the gel are carried to
the filter, where they are
permanently bound.
6.
Hybridization with labeled
probe. The filter is put into a
solution containing labeled
probe DNA. The probe binds
to DNA fragments
containing complementary
sequences.
7.
Visualize
fragments bound by
probe. Fluorescence
or autoradiography
(see BioSkills 7) is
used to find label.
Labeled
probe DNA
Filter
Gel
Sponge in
alkaline solution
SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET
DNA IN A LARGE COLLECTION OF DNA FRAGMENTS
Identifying Genes in Organisms
PCR
–
Polymerase Chain Reaction
Use: produce large amounts of DNA from a small
sample.
Basis: DNA replication in a test tube.
Procedure:
Isolate DNA or make cDNA from RNA
Add a solution with DNA polymerase, primers and
deoxynucleotides to produce copies of DNA.
dNTPs
Primers
5
2.
Denaturation
Heating leads to
denaturation of the
double
-
stranded DNA.
5
3
3
3
3
5
5
1.
Start with a solution
containing template DNA,
synthesized primers, and
an abundant supply of
the four dNTPs.
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE
MANY
IDENTICAL
COPIES
OF
A
SPECIFIC
GENE
5
4.
Extension
During incubation,
Taq
polymerase uses dNTPs to
synthesize complementary
DNA strand, starting at the
primer.
5
3
3
3
3
5
5
3.
Primer annealing
At cooler temperatures,
the primers bind to the
template DNA by
complementary base
pairing.
5
5
5
3
3
5
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE
MANY
IDENTICAL
COPIES
OF
A
SPECIFIC
GENE
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE
MANY
IDENTICAL
COPIES
OF
A
SPECIFIC
GENE
6.
Repeat cycle again,
up to 20
–
30 times, to
produce millions of
copies of template DNA.
5.
Repeat cycle
of three steps (2
–
4)
again, doubling the
copies of DNA.
Analyzing DNA
DNA sequencing
Use: identify gene base sequence from normal
organisms and mutations that cause disease.
Sequencing Procedure, fig 19.9:
Isolate specific gene (DNA)
Prepare 4 PCR solutions with labeled probes and
deoxynucleotides (dNTPs) + 1 dideoxynucleotide
(ddNTPs, have no OH group on 5’ end).
Analyzing DNA
Sequencing Procedure cont’d
When a ddNTP is incorporated in new DNA strands,
DNA synthesis stops.
Separate DNA fragments on a polyacrylamide gel.
Transfer bands to filter paper and expose to
photographic film. Band patterns indicate base
sequence of gene.
DIDEOXY SEQUENCING
3
3
5
5
Normal
dNTP
(extends
DNA strand)
ddNTP
(terminates
synthesis)
ddGTP’s
Template DNA
No OH
3
Labeled primer
Non
-
template DNA
1.
Incubate a large number of normal dNTP’s with a small
number of ddNTP’s (in this case starting with ddGTP’s),
template DNA, a primer for the target sequence, and DNA
polymerase.
2.
Collect DNA strands that are produced. Each
strand will end with a ddGTP (corresponding to
a C on the template strand).
5
3
5
ddCTP’s
ddATP’s
ddTTP’s
5
敮e
3
敮e
Smaller fragments
Larger fragments
Non
-
template DNA
5
5
3
3
Template DNA
3.
Repeat process three more
times using ddCTPs, ddATPs,
and ddTTPs, which will terminate
synthesis where G’s, T’s, and A’s
occur on the template strand,
respectively.
4.
Line up different
-
length strands by size using gel electro
-
phoresis to determine DNA sequence.
DNA
sequence
DIDEOXY SEQUENCING
Analyzing DNA
RFLP = restriction fragment length polymorphism.
Uses: forensic analysis, identifying patients with genetic
alterations that may cause disease.
Principle: DNA fragments of identical genes from
different individuals will not be exactly the same size
(point mutations, multiple copies, etc.).
Analyzing DNA
RFLP Procedure:
Cut DNA with several restriction endonucleases and
separate pieces using gel electrophoresis; compare band
patterns.
Band pattern (distribution of fragments by size) acts as a
DNA fingerprint
.
Biotechnology and you
Medical applications
Pharmaceuticals
–
“cheap” efficient production of
proteins used to fight disease. Ex. growth hormone &
insulin.
Gene therapy
–
introduction of a “healthy” gene into
an individual with a defective gene.
Identifying inherited diseases.
Identifying criminals
Biotechnology and you
Agricultural
Disease resistance
to crops and animals
Herbicide and insect resistance
to plants
Delay rotting
of fruits and vegetables
Introduce N
-
fixation ability
to plants
Genetic engineering of
“super”
plants and animals.
Risks?????
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