Download Cloning Genes

How to characterize a single piece of DNA
- Isolate a small fragment of DNA
- Insert DNA into plasmid (or phage vector)
-Transform recombinant DNA molecule into bacteria
-Amplify DNA by culturing transformed bacteria
-Select for transformants
-Use transformants for variety of purposes (e.g.expression studies,
sequencing, mutational analysis, etc.)
LE 20-2
Bacterium
Gene inserted into
plasmid
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
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
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
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
Restriction Enzymes Used to Make
Recombinant DNA
• Bacterial restriction enzymes
– cut DNA molecules at specific DNA sequences
called restriction sites
LE 20-3
Restriction site
DNA 5
3
3
5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Memorize
EcoRI restriction site
EcoRI
palindrome
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Catalyzes phosphodiester
bond between
5’ phosphate &
3’ hydroxyl group of sugar
Fragment from different
DNA molecule cut by the
same restriction enzyme
Ligation
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule
Do restriction digests and ligations always work?
What are the other possible undesirable outcomes?
What is a common strategy to select for transformed bacteria?
Grow bacteria on antibiotic: only plasmid carriers will survive
Clever way to select for recombinant clones
Plasmid
contains LacZ gene-->-galactosidase
X-gal (substrate:one
product is blue)
Blue colonies
Restriction site in LacZ gene
if insert DNA fragment
-galactosidase
X-gal
White colonies
LE 20-4_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
Different goals in creating recombinant clones
1. To examine/utilize the structure and function of a single
piece of DNA.
2. To package small pieces of an entire genome:
genomic DNA library
To have available all the sequences in the genome for
examination and use.
LE 20-6
DNA libraries created using plasmids and phage and bacterial hosts
or
Bacterial
clones
Foreign genome
cut up with
restriction
enzyme
Recombinant
plasmids
Recombinant
phage DNA
Plasmid library
Phage
clones
Phage library
Note: practical limit on the size of DNA cloned into a vectors
(plasmid: 5-10 kbp, phage: 45 kbp)
How to distinguish one DNA molecule from another?
Characterization of DNA by Size
Agarose Gel Electrophoresis
Digest DNA with restriction enzymes
Load DNA into wells of agarose gel
Apply electric current to fractionate
DNA fragments by size
In electric field with positive and negative poles, which pole
will DNA be attracted to? Why?
LE 20-8
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Shorter
molecules
Gel
Glass
plates
Anode
Longer
molecules
-DNA stained
with fluorescent
dye (ethidium bromide)
-DNA fluoresces upon exposure to
ultraviolet (UV) light
How would you determine whether a particular gene or
DNA sequence is present in your cloned DNA?
Southern Blot
LE 20-10
Restriction
fragments
DNA + restriction enzyme
I
II
III
Heavy
weight
Nitrocellulose
paper (blot)
Gel
Sponge
I Normal
-globin
allele
II Sickle-cell III Heterozygote
allele
Preparation of restriction fragments.
Labeled nucleic acid probe:
RNA or DNA
Radioactively
labeled probe
for -globin
gene is added
to solution in
a plastic bag
I
Paper
towels
Alkaline
solution
Gel electrophoresis.
Blotting.
Southern Blot Analysis
II
III
Probe hydrogenbonds to fragments
containing normal
or mutant -globin
I
II
Fragment from
sickle-cell
-globin allele
Paper blot
Hybridization with radioactive probe.
III
Film over
paper blot
Fragment from
normal -globin
allele
Autoradiography.
Why are globin DNA fragments different in size?
LE 20-9
Normal -globin allele
175 bp
Restriction enzyme
Ddel
201 bp
Ddel
Large fragment
Ddel
Ddel
Sickle-cell mutant -globin allele
376 bp
Ddel
Large fragment
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
-globin gene
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from normal
and sickle-cell alleles
Restriction Fragments Length Polymorphisms (RFLP)
- useful in detecting disease alleles
-forensics
to identify individuals
no two individuals are alike
(exception?)
LE 20-17
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)
Do the RFLPs
suggest the
defendant was
in contact with
the victim?
By themselves,
do RFLPS
prove she’s guilty
of assault?
Ch 20
Genomics and Molecular Techniques
• Characterization of entire genomes
• Human Genome Project (HPG):
•
ambitious goal to sequence the entire human genome
(initiated 1990; mostly complete 2003)
• Other genomes also sequenced
• Evolutionary relatedness of key interest
->sequence comparison
LE 20-11
Steps
in genome
mapping
Chromosome
bands
Cytogenetic map
(chromosome map)
Genes located
by FISH
Genetic (linkage)
mapping
Genetic
markers
Physical mapping
Overlapping
fragments
DNA sequencing
DNA Sequencing
• Short DNA fragments sequenced by dideoxy
chain-termination method
LE 20-12
DNA
(template strand)
5
Primer
3
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
DNA chain
terminators
5
DNA
polymerase
3
5
DNA (template
strand)
Labeled strands
3
Direction
of movement
of strands
Laser
Detector
3
• Other approach to genome sequencing:
– Shotgun method
• Sequence random fragments of DNA
• Computer program orders overlapping fragments
into single continuous sequence
LE 20-13
Cut the DNA from
many copies of an
entire chromosome
into overlapping fragments short enough
for sequencing
Clone the fragments
in plasmid or phage
vectors
Sequence each fragment
Order the
sequences into one
overall sequence
with computer
software
Can we learn important information from
the genome sequence?
• Genome organization
• Gene expression patterns in response to
- environmental change e.g.
pollution, global warming
-Development
embryogenesis->
senescence
-Disease/Health
Computer Analysis: Key Tool
Bioinformatics
-analysis and storage of biological data by
computing techniques
-key to management & analysis of huge
data sets
Example:
Identification of proteins coding sequences (ORF)
in genomes
agatactagcagctctttcgagcatcagcatcaccgatgcatcgatcacgcgctgtttg…
Think of a sequence feature that a program could search for
to identify ORFs.
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