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Transfer of genetic information by DNA
Gene technology & Molecular biology
Lecture 4
Recombinant DNA:cloning
Recombinant DNA
Generation of a recombinant DNA molecule
• Recombinant DNA technology provided
scientists with the ability to isolate,
sequence, and manipulate individual genes
from any type of cell.
• It has enabled detailed molecular studies of
the structure and function of eukaryotic
genes and genomes, and revolutionized our
understanding of cell biology.
A molecular tool box
• Enzymes
-restriction endonuclease
-ligase
-DNA polymerase
-reverse transcriptase (RT)
-kinase
-polynucleotide transferase
-phosphatase
• PCR
• In vitro mutagenesis
• Vectors
-plasmids
-phages (M13, λ)
-cosmides
-YACs
• Oligonucleotide synthesis -viruses
• Host cells
-primers
-probes
-bacteria
-gene assembly
-yeast/fungus/molds
-plant cells
• DNA sequencing
-insect cells
-Maxam-Gilbert
-mammalian cells
-Sanger
-pyrosequencing
A molecular tool box
• Transformation
• Enzymes
-heat-shock
-electroporation
-”gene gun”
-micro injection
-transfecion
-restriction endonuclease
-ligase
-DNA polymerase
-reverse transcriptase (RT)
-kinase
-polynucleotide transferase
-phosphatase
• Selection methods
-antibiotic resistance
-genetic complementation
• Screening methods
-genotypic
-phenotypic
• PCR
• In vitro mutagenesis
• Vectors
-plasmids
-phages (M13, λ)
-cosmides
-YACs
• Oligonucleotide synthesis -viruses
• Host cells
-primers
-probes
-bacteria
-gene assembly
-yeast/fungus/molds
-plant cells
• DNA sequencing
-insect cells
-Maxam-Gilbert
-mammalian cells
-Sanger
-pyrosequencing
• Transformation
-heat-shock
-electroporation
-”gene gun”
-micro injection
-transfecion
• Selection methods
-antibiotic resistance
-genetic complementation
• Screening methods
-genotypic
-phenotypic
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Restriction enzymes
Kloning - Expression
Enzymes
• Specific DNA sequences (4-8 bp) are recognized and cleaved by restriction
endonucleases (RE)
• Recognition sequence (RS) is often palindromic (can be read from both ends)
• Is part of the defense system against foreign DNA; > hundreds of different RE
• Specific methylases methylates certain bases in the RS, whereby the bacterium’s own
DNA becomes protected from cleavage
EcoRV
EcoRI
PstI
GATATC
CTATAG
GAATTC
CTTAAG
CTGCAG
GACGTC
GAT
CTA
ATC
TAG
”blunt end”
Recognition sites of common RE
G
AATTC
CTGCA
G
G
G
ACGTC
CTTAA
”sticky ends”
Restriction map
EcoRI recognizes the sequence GAATTC.
This sequence is present at five sites in DNA of the bacteriophage λ, so EcoRI digests λ DNA into six
fragments ranging from 3.6 to 21.2 kilobases long.
Restriction maps of λ and adenovirus DNAs
Restriction enzymes
• For larger DNA molecules such as cellular genomes, restriction
endonuclease digestion alone does not provide sufficient resolution.
• For example, the human genome would yield more than 500,000 EcoRI
fragments.
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Restriction enzymes
Ligase
• Ligases recreate phosphodiesterbonds by joining the 5’-phosphate group to the
free 3’-hydroxyl group. A common enzyme is the T4-DNA ligase.
• Ligases are used to ”glue” DNA fragments to each other.
G-OH P-GATCT
CCTAG-P HO-A
G-OH P-GATCC
CCTAG-P HO-G
+ T4-DNA ligase, ATP
+ T4-DNA ligase, ATP
G GATCT
CCTAG A
GGATCC
CCTAGG
can be re-cleaved by BamHI
(5’-GˇGATCC-3’)
Ligase reaction
cannot be re-cleaved by BamHI
(5’-GˇGATCC-3’)
DNA polymerases
• DNA polymerases incorporates deoxynucleotides (dNTPs) to a
growing DNA chain; the dNTPs are added to the free 3’-OH group
using the opposite strand as template. Some frequently used
polymerases are DNA polymerase I, T7-DNA polymerase, and Taqpolymerase.
• They are used for several things, for example:
- generation of blunt-ends
- in DNA sequencing reactions
G
CCATG
Reverse transcription and retrovirus replication
+ dNTPs, polymerase
GGTAC
CCATG
Reverse transcriptase (RT)
• Reverse transcriptase (RT) synthesizes a complementary DNA
chain from a RNA template. Retroviruses (e.g., HIV) use this
mechanism to make a DNA copy of it’s RNA genome.
• Can be used to our advantage:
- in cDNA synthesis
mRNA 5’Cap
AAAAA 3’
TTTTT 5’
reverse transcriptase
+ dNTPs
mRNA 5’Cap
cDNA
3’
AAAAA 3’
TTTTT 5’
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Terminal transferase
Phosphatases och kinases
• Terminal transferase adds nucleotides to the 3’-end of DNA
chains.
• Used, among other things, for generation of cohesive ends from
blunt-end fragments and also at one step in cDNA synthesis
5’
3’
+
3’
dATP
5’
dTTP
terminal transferase
3’
AA…AA-OH
5’
HO-TT…TT
3’
5’
• Phosphatases remove free 5’-phosphate groups from DNA
- used to minimize the incidence of self-ligated vector in DNA
cloning
- commonly used phosphatases are the shrimp- and bovine alkaline
phosphatase
• Kinases adds phosphate groups to the 5’-end of DNA
- used to phosphorylate synthetic DNA (e.g., linkers) before ligation
- polynucleotidekinase (PNK)
ligation
5’
AA…AA
3’
3’
TT…TT
5’
Kloning - Expression
Vectors
Cloning vectors: desired properties
• small
• ”universal”; should work in different organisms
• easy to isolate from the host organism
• easy to detect and select
• multiple copies (is usually advantageous)
• several unique RE localized to a specific region (mcs)
• convenient method for detection of cloned DNA
Plasmids
Plasmid preparation
• Plasmids are circular extrachromosomal DNA
• Used as vectors = carriers of ”foreign” DNA
- used in cloning and recombinant protein expression
- insert size ≤ 5 kb
• Plasmids usually contain:
- ori (origin of replication)
- gene/s for selection (often antibiotic resistance genes; bla, cat)
- multiple cloning site (mcs)
- sometimes a gene that allows for screening (e.g., lacZ for blue-white
screening)
• Different plasmid-types have different ”copy nr”
• Plasmids belong to different incompatibility groups; two plasmids belonging
to the same group cannot be stably propagated simultaneously within a single
cell.
Figure 8-40 Molecular Biology of the Cell (© Garland Science 2008)
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Bacteriophage λ
Bacteriophage λ-life cycle
Figure 5-78 Molecular Biology of the Cell (© Garland Science 2008)
Bacteriophage λ
In vitro packaging:bacteriophage λ
• Bacteriophage λ is a virus that infects bacteria (E. coli).
- a lytic and a lysogenic phase (prophage) in the life cycle.
- genome size approx. 45 kb; a central region of 15 kb is not
essential for replication.
- have complementary single stranded cohesive ends (COS-sites);
used by the phage to make concatamers of it’s genome when the
DNA is packed into phage particles.
• the λ genome has been modified to work better as a vector;
unique sites that flank the central region have been introduced to
simplify DNA cloning/replacement.
- inserts must have a certain size (approx. 15 kb), or else no
infectious phage particles can be formed.
• λ used for cloning of genomic fragments, since plasmids are not
suitable for cloning of larger DNA fragments.
Cosmids
• Cosmids are plasmid-phage λ hybrids. They contain:
- ori
- gene for antibiotic resistance (e.g., Tetr)
- cos-sites
- cloning sites
• Cosmids are used for cloning of genomic DNA fragments larger
than 15 kb (this is the limit of what phage λ can harbor). Cosmids
can accommodate an insert size of approx. 45 kb.
cos
vector DNA (cosmid)
Biotechnology: Applying the genetic revolution, Figure 3.17
Yeast artificial chromosome (YAC)
• YACs contain:
- autonomously replicating sequence (ARS) from yeast
chromosome
- centromer (CEN); ensures stable and even distribution of the
YACs between mother and daughter cells
- two telomers; constitute chromosome ends and enables
replication of the YACs as small linear chromosomes.
- selectable gene markers, e.g., LEU2 that complement Leu
negative strains.
- cloning sites
• YACs used for cloning of very large genomic fragments (1002000 kb)
antibiotic resistance
gene
genomic DNA
telomer
LEU2
ARS
CEN
jäst DNA
telomer
50 kb
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Various cloning vectors
Vectors for cloning large DNA fragments
Biotechnology: Applying the genetic
revolution, Figure 3.16
Host cells
Kloning - Expression
Host cells
Different vectors are used to introduce recombinant DNA
in various types of host cells, for example:
• Baceria
•
•
•
•
Kloning - Expression
- E. coli the most common
- High expression levels
- Relatively easy to scale-up the production process.
Yeast
Insect cells
Eukaryotic cells
Mammalian cells
Plant cells
Post-translational modifications
(e.g., glycosylations) possible
Heat shock transformation
Transformation
methods
Biotechnology: Applying the genetic revolution, Figure 3.18
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Electroporation
Kloning - Expression
Selection methods
Acta Physiol Scand. 2003 Apr;177(4):437-47
Issuses to consider:
1. Cell size
2. Temperature
3. Post-pulse manipulation
4. Composition of electrodes and pulsing medium
Antibiotic resistance
Commonly used antibiotics in molecular
biology:
-
ampicillin
Inhibits cell wall synthesis
carbenicillin
chloramphenicol
Inhibits protein synthesis
tetracyclin
kanamycin
Transform bacteria with
ligated plasmid
Kloning - Expression
Cloned gene
Gene for
antibioticresistance
Spread bacteria and grow on
solid growth media (agar)
supplemented with antibiotics
Screening methods
Plasmid
Only bacteria containing the
plasmid with the antibiotic
resistance gene survive and
form colonies
Genotypic screening
• Probe hybridization
– known nucleotide sequence (gene sequence)
• 15 nt long probe
– unknown nucleotide sequence (gene sequence)
• “guessmer”probe, approx. 50 nt long (unique)
longer due to risk of mis-match
• degenerate probe, approx. 18 nt long
based on amino acid sequence (6 residues long)
Phenotypic screening
• Activity based
– e.g., protease activity (subtilisin)
• grow on casein media and a clear zone (“halo”) will
appear around proteolytically active clones
• Immuno based
– antibody specific for the protein of interest
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Blue-white screening
Kloning - Expression
EcoRI site
LacZ
AmpR
Cloning of a DNA fragment in
the EcoRI site in LacZ (encodes
the enzyme β-galactosidase)
An example of gene
cloning
- only the bacteria that harbor the
plasmid are resistant to ampicillin and
can grow
Transform bacteria and
grow on agar plates
supplemented w
ampicillin and X-gal
- bacteria w/o cloned fragment have a
functional β-galactosidase gene and
convert the color-less substrate X-gal
to a blue-colored product
H
N
OH
OH
H
OH
O
Br
Cl
H
H
H
OH
- in bacteria w a cloned fragment the
β-galaktosidase gene is destroyed and
therefore only give rise to white
colonies
O
H
X-gal
Cloning of a prokaryotic gene
• Example: Subtilisin
•
•
•
•
from Bacillus subtilis
protease
gene size, approx. 1000 bp
used in washing powders
Create a gene library
BamHI
EcoRI
BamHI
Bacterial plasmid
GATCC
G
G TAG
CC
G
CTTAA
C
TT G
AA
create a gene library (genomic DNA)
selection and genotypic screening
phenotypic screening
sequence verification
clone into an expression vector
activity studies/protein engineering
EcoRI
Chromosome
• Sub tasks
•
•
•
•
•
•
EcoRI
DNA ligase
Transformation
Gene library
• Chromosome digested w RE (“4-cutter”)
• (4x4x4x4=256 bp) [e.g., Sau3AI, ^GATC^]
• Partial digestion
– Large randomly digested gene fragments (~2500 bp)
• Plasmid cleaved w compatible RE (“6-cutter”)
• Matching overlaps [e.g., BamHI, G^GATC^C]
• Ligation (Phosphatase treated plasmid)
• Transformation (CaCl2, Electroporation)
• Selection
• Colonies with recombinant plasmid
Gene library
Creating a DNA Library:
Genomic DNA from the chosen organism is first
partially digested with a restriction enzyme that
recognizes a four base-pair sequence. Partial
digestions are preferred because some of the
restriction enzyme sites are not cut, and larger
fragments are generated. If every recognition site
were cut by the restriction enzyme, then the genomic
DNA would not contain many whole genes. The
genomic fragments are cloned into an appropriate
vector, and transformed and maintained in bacteria.
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Screening a recombinant library by hybridization
Cloning of a eukaryotic gene
• Often mRNA as source
• Sub tasks
• Create a gene library
– mRNA purification
– cDNA synthesis (S1-method, RNaseH-method)
– Ligation into a vector (phage or plasmid)
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•
•
•
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Selection and genotypic screening
Phenotypic screening
Sequence verification
Expression vector (choice of host!)
Activity studies/protein engineering
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