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Molecular Biology
Lecture 3
Chapter 4
Molecular Cloning
Methods
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Lecture outline
• Gene cloning
• Restriction endonucleases
• Plasmids
pBR322
pUC
• Selection
4-2
Gene Cloning
• Introduce a foreign gene or piece of DNA
into a suitable vector and inserting the
recombinant molecule into a bacterial host
• Cloning can also be done in eukaryotic
cells such as yeast
• One can then produce large quantities of
the gene or piece of DNA in pure form
4-3
The Role of Restriction
Endonucleases
• Restriction endonucleases, first
discovered in the late 1960s, are named
for preventing invasion by foreign DNA by
cutting it into pieces
• These enzymes cut at sites within the
foreign DNA instead of chewing from the
ends
• By cutting DNA at specific sites they
function as finely honed molecular knives
4-4
Restriction-Modification System
• What prevents these enzymes
from cutting up the host DNA?
– They are paired with methylases
– Theses enzymes recognize, and
methylate the same site
• The sequence specific
methylase and restriction
endonuclease are called a
restriction-modification system,
R-M system
• Methylation protects DNA, after
replication (the parental strand is
already methylated)
4-5
Restriction Endonuclease
Specificity
• A 6-bp cutter will yield DNA fragments averaging
4000-bp or 4 kilobases (4kb) in length
e.g. EcoR1 recognize the sequence
GAATTC
CTTAAG
Probability of finding the sequence
1/.25 X .25 X .25 X .25 X .25 X .25 = 4096
4-6
Restriction Endonuclease
Specificity
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY at
that sequence
– These enzymes can
recognize 4-bp, 6-bp, 8-bp
sequences
– The frequency of cuts
lessens when the
recognition sequence is
longer
4-7
Use of Restriction Endonucleases
• 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
4-8
Use of Restriction Endonucleases
• Example EcoR1
– This enzyme leaves single-stranded
overhangs, called sticky ends that can basepair together
GAATTC
CTTAAG
+ EcoR1 =
G
CTTAA
and
AATTC
G
4-9
Use of Restriction Endonucleases
• Making a recombinant DNA molecule
+
in presence of DNA ligase and ATP
GAATTC
CTTAAG
DNA ligase will synthesize the phosphodiester bonds
4-10
Summary
• Restriction endonucleases recognize
specific sequences in DNA molecules and
make cuts in both strands
• This allows very specific cutting of DNAs
• The cuts in the two strands are frequently
staggered, so restriction enzymes can
create sticky ends that help to link together
2 DNAs to form a recombinant DNA in vitro
4-11
Vectors
• Vectors function as DNA carriers to allow
replication of recombinant DNAs
• Typical experiment uses 1 vector plus a
piece of foreign DNA
– Foreign DNA has no origin of replication, the
site where DNA replication begins
– Depends on the vector for its replication
• There are 2 major classes of vectors:
– Plasmids
– Phages (not covered in this course)
4-12
First cloning Experiment Using
Restriction Endonuclease
• An early experiment used EcoRI to
cut 2 plasmids
Small circular pieces of DNA independent of
the host chromosome
• Each plasmid had 1 site for EcoRI
– Cutting converted circular plasmids
into linear DNA with the same sticky
ends
– The ends base pair
• Some ends re-close
• Others join the 2 pieces
• DNA ligase joins 2 pieces with
covalent bonds
4-13
Plasmids As Vectors
• pBR plasmids were developed early but
are rarely used today
• pUC series is similar to pBR
– 40% of the DNA, including tetracycline
resistance has been deleted
– Cloning sites are clustered together into one
area called the multiple cloning site (MCS)
4-14
pBR322 Plasmid
• 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
4-15
pBR322 Cloning
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
4-16
Bacterial Transformation
• Traditional method involves incubating
bacterial cells in concentrated calcium salt
solution
– The solution makes the cell membrane leaky,
permeable to the plasmid DNA
• Newer method uses high voltage to drive
the DNA into the cells in process called
electroporation
4-17
Screening Transformants
• Transformation produces bacteria with:
– Religated plasmid
– Religated insert
– Recombinants
• Identify the recombinants using the antibiotic
resistance
– Grow cells with tetracycline so only cells with plasmid
grow, not foreign DNA only
– Next, grow copies of the original colonies with
ampicillin which kills cells with plasmid including
foreign DNA
4-18
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
4-19
Directional Cloning
• Cut a plasmid with 2 restriction enzymes
• 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
4-20
pUC and b-galactosidase
Newer pUC plasmid
4-21
pUC and b-galactosidase
Newer pUC plasmid
lac promoter
O
MCS
lacZ (a-peptide)
-Induced by lactose or IPTG
-Under the control of the lac repressor
-The MCS is a cluster of sequences recognized by restriction
endonucleases
4-22
pUC and b-galactosidase
Clones with foreign DNA in the MCS disrupt the
ability of the cells to make b-galactosidase
b-galactosidase is encoded by the lacZ gene of the lac
operon
b-galactosidase cleaves lactose and can also cleave the
synthetic substrate X-gal.
Cleaved X-gal gives a blue coloration. You can monitor
the activity of b-galactosidase by looking at the blue
coloration
4-23
pUC and b-galactosidase
a-complementation
Plasmid contains part of the lacZ gene coding for the Nterminal extremity of the b-galactosidase enzyme.
When expressed in E. coli lacZ strain = no activity
Host bacterial strain contains a truncated lacZ gene
encoding a polypeptide missing the N-terminal extremity
When expressed in E. coli = no activity
4-24
pUC and b-galactosidase
a-complementation
When the plasmid is introduced in the bacterial strain
containing the truncated enzyme, activity is recovered.
The two partial gene products can cooperate to form an
active enzyme
(model on the blackboard)
4-25
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 (but requires replica plating)
• pUC has
– Ampicillin resistance gene
– MCS that interrupts a b-galactosidase gene
• MCS facilitates directional cloning into 2 different
restriction sites
4-26
4-27