Transcript 2 - cellbiochem.ca

Recombinant DNA Technology
CHMI 4226 E
Week 2
12 January 2009
Toolbox part 2.
Plasmids, DNA cloning 101, DNA
sequencing
E.R. Gauthier, Ph.D.
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Plasmids
• Plasmids:
– Extrachromosomal (episomal) circular DNA molecules
– Minimal features:
• Origin of replication: allow the plasmid to replicate
autonomously
• Antibiotic resistance gene: allow for selection for bacterial
cells that have taken up the vector
• Multiple cloning site (MCS): a small region of the plasmid
engineered to contain the cleavage sites for a limited number
of restriction enzymes
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Plasmids – origin of replication
• Origin of replication (Ori): small DNA sequences allowing the
bacterial DNA polymerase to bind and initiate DNA replication;
• Several Ori can be found. ColE1 is the most frequently encountered;
• Some Ori allow the plasmid to replicate frequently (high copy
number plasmids – up to 100 copies per cell); others allow only a
low rate of replication initiation (low copy number plasmids – only
a few copies per cell)
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Plasmids – selection genes
• Genes which encode for proteins affording resistance to a specific
antiibotic;
• Most frequently encountered:
– Ampr: resistance to ampicillin
– Kanr: resistance to kanamycin
– Tetr: resistance to tetracyclin
• Bacteria possessing a plasmid with the Ampr gene will survive when
plated onto a media containing ampicillin;
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Plasmids – multiple cloning sites
• MCS: a limited region on the plasmid which has been
engineered to contain unique cleavage sites for a
selected number of restriction enzymes;
• RE cleavage sites in the MCS are not found anywhere
else on the plasmid.
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Plasmids – other features
• Promoters for viral RNA polymerases Sp6, T7 or T3
– Allows for in-vitro trasncription experiments
• Origin of replication of phage F1:
– Enables the production of single-stranded plasmids
• Genes encoding supressor tRNAs
• Reporter genes:
– Allows for the easy identification of bacterial cells with desired
features
– E.g.: Lac z (encodes b-galactosidase)
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Plasmids – SupF selection gene
• SupF:
– Suppressor tRNA
– Inserts an amino acid
(Glu) at the UAA stop
codon
– Allows the survival of
bacteria with a P3
episome : a plasmid
containing the Ampr
and Tetr genes both
interrupted with a UAA
stop codon.
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Plasmids – pBR322
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Plasmids - pBluescript
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Plasmids – shuttle vectors
• Shuttle vectors can
be used in at least
two different
organisms:
–
–
–
–
–
Bacteria (mandatory)
Yeast
Insects
plants
mammals
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Plasmids – shuttle vectors
• Bacteria/Yeast shuttle
vector;
• Leu2:
– Selection marker for growth
in yeast
– Encodes for a gene which
allows for the synthesis of
the amino acid leucine in a
yeast strain which cannot
produce Leu on its own
(auxotrophic)
– Yeast with this vector can
be grown on media devoid
fo leucine.
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DNA cloning
• Why cloning DNA?
– To produce greater amounts of YFG
– To characterise the properties of YFG
• sequencing
• Mutagenesis
– To express YFP in vitro or in a living organism
• production of recombinant insulin: much better than insulin
purified from blood, which can be contaminated with viruses
(hepatitis C, HIV) or other nice things (prions)
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DNA cloning – basic steps
• 1. cut DNA of interest and plasmid with appropriate
restriction enzyme
• 2. mix together and seal free ends with DNA ligase (ligation)
• 3. Insert DNA in bacteria (transformation)
– Requires transformation-competant bacteria
• 4. Select antibiotic-resistant bacteria and search for cells
having the recombinant plasmid.
• 5. Confirm cloning
– restriction enzyme digest
– DNA sequencing
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DNA cloning – basic steps
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DNA cloning
• Treating vector with alcaline phosphatase
– reduces background of bacteria with plasmid not containing the
insert.
• Blunt-ends can be used:
– a greater insert DNA/plasmid ratio must be used: 10/1 instead of
the usual 3/1
– Much, much less efficient than cloning with protruding ends
• The ends of your DNA fragment can be modified with
Klenow enzyme to accomodate the available RE sites in
the vector (and vice-versa).
• Adaptors and linkers can be used to facilitate cloning.
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Adaptors and linkers
Adaptors
Linkers
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Assignment #2
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Use of b-gal
- Bacteria used in genetic engineering
express a portion of b-gal missing the Nterminal 50 amino acids (called the
omega [w] fragment);
-Several vectors carry the missing Nterminal 50 amino acids (a fragment) of bgal in the MCS;
- Only the bacteria having both the a and
w portion of b-gal can express a functional
enzyme (a complementation)
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Use of b-gal
• If a DNA fragment is inserted
into the MCS of pBluescript, it
interrupts the b-gal a fragment,
making it inactive;
• SO: bacteria with a
recombinant plasmid will not
express a functional b-gal, and
will turn white upon staining
wity X-gal;
• However, bacteria with a
plasmid which does not
contain the DNA fragment will
produce the a fragment, will
have a functional b-gal
enzyme, and will turn blue
upon staining with X-gal.
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DNA cloning – plasmid purification
from bacteria
Culture 1
bacterial colony
into liquid media
.....
.
Recover bacterial
cells by
centrifugation
.....
.
1) Resuspend
cells in glucosebased buffer
3) Precipitate
bacterial
chromosome and
proteins with
acidic buffer and
centrifuge
E.R. Gauthier, Ph.D.
2) Lyse bacteria
with SDS/NaOH
buffer
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Extract supernatant
with basic (pH 8)
phenol - solubilizes
contaminating
proteins
Extract with
chloroform – to
get rid of the
phenol
Precipitate plasmid
DNA with ethanol
Dry pellet,
resuspend in
buffer, digest with
restriction enzyme
and analyse on
agarose gel
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DNA cloning – identification of
recombinant plasmids
• Three important tests
have to be done:
– Cut with RE to see if the
insert is of the correct
size;
– Cut with RE to confirm the
identity of the fragment
(usually choose RE that cut
inside the fragment)
– Sequence the insert.
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DNA sequencing
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DNA sequencing
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DNA sequencing
3’
A
G
T
C
G
C
A
C
T
A
G
T
G
C
A
T
A
G
5’
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DNA sequencing
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DNA sequencing
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What to do with the data?
• 1. identification (did I clone the piece of
DNA I wanted)
• 2. Is it complete, or do did I clone only a
portion of it (happens more often than you
think…)?
• Are there any mutations (happens a
lot…)?
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BLAST search
• Blast: algorithm that allows you to quickly
identify sequences that are similar to the
DNA of interest;
• Blastp: similarity searches with amino acid
sequences
• Blastn: similarity searches with nucleotide
sequences
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BLAST search
QUERY sequence(s)
BLAST results
BLAST program
BLAST
database
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BLAST search
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BLAST search
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BLAST search
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BLAST search
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BLAST search
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BLAST search
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BLAST search
• E value: indicate the
odds that the hit is
attributable to chance
only.
– The lower the E
value, the better the
odds that it is a real
match.
• G: link to Entrez Gene
• U: link to sequencerelated info (expression
profile, chromosomal
location, orthologs)
• E: link to GEO (Gene
Expression Omnibus):
database of gene
expression profiles.
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BLAST search
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BLAST search
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Translation of sequencing data
• Allows you you determine if the sequence
you isolated contains the entire open
reading frame.
– Open reading frame (ORF):
• the nucleotide sequence coding for a protein;
• Starts with a AUG codon, and stops with one of the
3 stop codons (UAA, UAG, UGA).
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BLAST search
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Translation of sequencing data
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Translation of sequencing data
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Translation of sequencing data
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Translation of sequencing data
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Translation of sequencing data
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Translation of sequencing data
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Blastp result
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Assignment #3!
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