Transcript Chapter 3 part I

Recombinant DNA Technology
Recombinant DNA
• Protocols that transfer genetic information
(DNA) from one organism to another.
• Gene cloning links eukaryotic genes to
small bacterial or phage DNAs and
inserting these recombinant molecules into
bacterial hosts.
• One can then produce large quantities of
these genes in pure form.
Recombinant DNA - Gene Cloning
 DNA from a source
organism is cleaved
with restriction
endonuclease and
inserted into a cloning
vector.
 The recombinant vector
is introduced into a host
cell.
 Recombinant cells are
identified and grown.
Five Basic Steps in Gene Cloning
• The first is to choose the appropriate DNA to
be cloned, genomic or cDNA.
• Produce a collection of DNA fragments of
size suitable for inserting into appropriate
vectors.
• Insert DNA fragments into the vector using
DNA ligase (DNA ligation.)
• Introduce DNA fragments into a population of
bacteria (transformation.)
• Select the colonies containing desired
sequence from the “library.”
Restriction Enzymes
 Restriction enzymes (restriction
endonucleases) cut double-stranded DNA into
smaller pieces.
 Bacteria use these as defense against DNA from
bacteriophage.
 DNA is cut between the 3′ hydroxyl group of one
nucleotide and the 5′ phosphate group of the
next - restriction digestion.
Restriction Enzymes
• Restriction enzymes do not cut
bacteria’s own DNA because the
recognition sequences are modified.
• Methylases add methyl groups after
replication; makes sequence
unrecognizable by restriction enzyme.
Restriction Enzymes
• Bacterial restriction enzymes can be
isolated from cells.
• DNA from any organism will be cut
wherever the recognition site occurs.
• EcoRI (from E. coli) cuts DNA at this
sequence: GAATTC
Restriction Enzymes
The sequence is palindromic—it reads
the same in both directions from the 5′
end.
EcoRI occurs about once every four
genes in prokaryotes. DNA can be
chopped into small pieces containing a
few genes.
Restriction Enzymes
Restriction endonucleases are named using
the 1st three letters of their name from the
Latin name of their source microorganism
Hind III
– First letter is from the genus H from Haemophilus
– Next two letters are the 1st two letters of the species name in
from influenzae
– Sometimes the strain designation is included
“d” from strain Rd
– If microorganism produces only 1 restriction enzyme, end the
name with Roman numeral I Hind I
– If more than one restriction enzyme is produced, the others are
numbered sequentially II, III, IV, etc.
Restriction Enzymes
• These enzymes can recognize 4-bp, 6-bp,
8-bp sequences
• The frequency of cuts lessens when the
recognition sequence is longer
• A 6-bp cutter will yield DNA fragments
averaging ~ 4000-bp or 4 kilobases (4kb)
in length
Restriction Enzymes
• Many restriction endonucleases make
staggered cuts in the 2 DNA strands
– This leaves single-stranded overhangs, called
sticky ends that can base-pair together briefly
– This makes joining 2 different DNA molecules
together much easier
• Staggered cuts occur when the recognition
sequence usually displays twofold
symmetry, palindromes
Staggered Cleavage
Blunt-end Cleavage
Restriction Enzymes
• Heteroschizomers (isochizomers)
recognize the same DNA sequence but
are from different organisms.
• Restriction nuclease that recognize the
same DNA sequence but use a different
cutting site – they are also called
neoschizomers.
• Restriction nuclease that produce the
same nucleotide extension are called
isocaudomers.
Restriction Enzymes
• These enzymes cut DNA strands reproducibly
in the same place, which is extremely useful in
gene analysis
• Isoschizomers cleave a sequence only if the
cytosines of the recognition site are not
methylated whereas another will cut the same
sequence if these cytosines are methylated.
• For example, HpaII cuts only nonmethylated
CCGG sites, and MspI cuts this sequence
regardless of cytosine methylation.
Neoschizomers
Annealing of complementary extensions
Gel Electrophoresis
• After DNA is cut, fragments of different
sizes can be separated by gel
electrophoresis.
• Mixture of fragments is place on a well
in a porous gel. An electric field is
applied across the gel. Negatively
charged DNA fragments move towards
positive end.
• Smaller fragments move faster than
larger ones.
Restriction sites mapping
Restriction sites mapping
Plasmid Cloning Vectors
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Plasmids are small, circular DNA molecules that are
maintained as independent extrachromosomal entities.
F plasmids carry information for their own transfer from
one cell to another.
R plasmids encode resistant to antibiotics.
Cryptic plasmids have no apparent functional coding
genes.
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Plasmids can easily incorporate foreign DNA.
Plasmids are readily taken up by bacterial cells.
Plasmids then act as vectors, DNA carriers that
move genes from one cell to another.
Each plasmid has a sequence that functions as an
origin of DNA replication.
Vectors to Carry DNA Sequences
A vector should have four characteristics:
• Ability to replicate independently of the
host cell
• A recognition sequence for a restriction
enzyme (cloning site)
• One or more selectable/reporter genes
• Small size in comparison with host’s
chromosomes
Vectors to Carry DNA Sequences
Plasmids have all these characteristics.
• Plasmids are small, many have only
one restriction site.
• Genes for antibiotic resistance can be
used as reporter genes.
• And they have an origin of replication
and can replicate independently.
Plasmid Cloning vector pBR322
• pBR322 illustrates cloning methods simply
– Resistance for 2 antibiotics
• Tetracycline
• Ampicillin
– Origin of replication between the 2
resistance genes
– Only 1 site for several restriction enzymes
Cloning using pBR322
Clone a foreign DNA into
the PstI site of pBR322
 Cut the vector to
generate the sticky ends
 Cut foreign DNA with PstI
also – compatible ends
 Combine vector and
foreign DNA with DNA
ligase to seal sticky ends
 Now transform the
plasmid into E. coli
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Cloning using pBR322
If new DNA is inserted at that PstI
restriction site, it inactivates the gene
for ampicillin resistance.
Plasmid then has gene for tetracyclin
resistance, but not for ampicillin. This
can be used to select for host cells with
new DNA.
Cloning using pBR322
The cleaved plasmid DNA
preparation is treated with
enzyme alkaline
phosphatase to remove
the 5’ phosphate groups
from the linearized plasmid
DNA.
Transformation and selection
 Traditional method involves incubating bacterial cells
in concentrated calcium salt solution
 The solution makes the cell membrane leaky,
permeable to the plasmid DNA – competent cells.
 Newer method uses high voltage to drive the DNA
into the cells in process called electroporation
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Transformation and selection
 A natural transformation process often entails
 The binding of double-stranded DNA to component of
the cell wall.
 Entry of the DNA into an inner periplasm.
 Transmission of one strand into the cytoplasm while
the other one is degraded.
 If the DNA is linear molecule, integration into host
chromosome.
 If the introduced DNA is a plasmid, it is maintained in
the cytoplasm after the second strand is synthesized.
www.ncbi.nlm.nih.gov/bookshelf/picrender.fcgi...
Transformation and selection
 Transformation produces bacteria with:
 Religated plasmid
 Religated insert
 Recombinants plasmid
 Identify the recombinants using the antibiotic
resistance
 Grow cells with tetracycline so only cells with plasmid
grow, not foreign DNA only (religated insert)
 Next, grow copies of the original colonies with
ampicillin which kills cells with plasmid including
foreign DNA (Figure 3.11)
Clone a foreign DNA
into the BamHI site
 Cells contain no
plasmid are sensitive
to both Amp and Tet.
 Cells contains intact
plasmids are resistant
to both.
 Cells contains
inserted plasmids are
resistant to Tet but
sensitive to Amp.
Screening with replica plating
 Replica plating transfers
clone copies from original
tetracycline plate to a plate
containing ampicillin
 A sterile velvet transfer tool
can be used to transfer
copies of the original
colonies
 Desired colonies are those
that do NOT grow on the
new ampicillin plate
pUC and β - galactosidase
Newer pUC plasmids have:
 Ampicillin resistance gene
 Multiple cloning site inserted into the gene lacZ’ coding
for the enzyme β-galactosidase
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Clones with foreign DNA in the MCS disrupt the ability of the
cells to make β-galactosidase
Plate on media with a β-galactosidase indicator (X-gal) and
clones with intact β-galactosidase enzyme will produce blue
colonies
Colorless (desirable) colonies should contain the plasmid
with foreign DNA
pUC and β - galactosidase
Directional cloning
 Cut a plasmid with 2 restriction enzymes from the
MCS
 Clone in a piece of foreign DNA with 1 sticky end
recognizing each enzyme
 The insert DNA is placed into the vector in only 1
orientation
 Vector religation is also prevented as the two
restriction sites are incompatible
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• Digest plasmid using
EcoRI and XhoI
• Ligate cDNA into
digested plasmids
• Transformation –
introduce recombinant
plasmids into bacterial host
cells.
• Select transformants
using blue-white
screening.
Summary
 First generation plasmid cloning vectors include
pBR322 and the pUC plasmids
 pBR322 has
 2 antibiotic resistance genes
 Variety of unique restriction sites for inserting foreign
DNA
 Most of these sites interrupt antibiotic resistance,
making screening straightforward
 pUC has
 Ampicillin resistance gene
 MCS that interrupts a β-galactosidase gene
 MCS facilitates directional cloning into 2 different
restriction sites
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Some antibiotics commonly used
as selective agents
Vector backbone exchange: SfiIx-SfiIy
• Other bacteria, such as Bacillus subtilis and
Agrobacterium tumefaciens, often act as the final
host.
• Cloning vectors that function in E. coli maybe
provided with a second origin of replication.
• In addition, a number of plasmid vectors have
been constructed with a single broad-host-range
origin of DNA replication so they can be used with
a variety of microorganisms.
Vector backbone exchange: SfiIx-SfiIy
• The size of vector increases because of the
additional sequence resulting in decreasing the
amount of DNA that can be inserted.
• Shuttle vectors are not efficiently propagated in
the host cell.
• Broad-host-range cloning vectors can be
unstable and can be lost from a preferred host
cells.
• The chimeric vectors are engineered.
Vector backbone exchange: SfiIx-SfiIy
Vector backbone exchange: SfiIx-SfiIy
• Cell without any plasmid and those with plasmids
without chloramphenicol resistance gene cannot
grow in the presence of chloramphenicol.
• Plasmids that do not carry the origin of replication
or that contain E. coli origin of replication will not
be replicated in the host cell.
• Only cells that carry the chimeric plasmid with the
origin of replication in the host cell and
chlorampenicol resistance gene will be selected.
Making a genomic library
• The first is to choose the appropriate DNA to
be cloned, genomic or cDNA.
• Produce a collection of DNA fragments of
size suitable for inserting into appropriate
vectors – partial digestion.
• Insert DNA fragments into the vector using
DNA ligase (DNA ligation.)
• Introduce DNA fragments into a population of
bacteria (transformation.)
• Select the colonies containing desired
sequence from the “library.”
Making a genomic library
Making a genomic library
Vectors to Carry DNA Sequences
Plasmids can be used for genes of
10,000 bp or less. Most eukaryote
genes are larger than this.
Viruses can be used as vectors—e.g.,
bacteriophage. The genes that cause
host cell to lyse can be cut out and
replaced with other DNA.
Vectors to Carry DNA Sequences
Bacterial plasmids don’t work for yeasts
because the origins of replication use
different sequences.
A yeast artificial chromosome (YAC)
has been created: contains yeast origin
of replication, plus yeast centromere
and telomere sequences.
Also contains artificial restriction sites
and reporter genes
How many clones do we need?
• The sum of the inserted DNA in the clones of
the library should be three or more times the
amount of the DNA in the genome.
• For example, if a genome has 4 x 106 bp and
the average size of an insert is 1,000 bp,
then 12,000 clones are required for threefold
coverage.
• For human genome (3.3 x 109 bp), about
80,000 BAC clones that have an average
insert size of 150,000 bp compose a library
with fourfold coverage.