Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver

Chapter 4
Molecular Cloning
Methods

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 4.1 Gene Cloning
• 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

4-2
 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
4-3
 Naming Restriction
Endonucleases
Restriction endonucleases are named using the 1 st
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.
4-4
 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
4-5
 Restriction Enzyme
Terminology
• A 6-bp cutter will yield DNA fragments
averaging 4000-bp or 4 kilobases (4kb) in
length
• Heteroschizomers recognize the same
DNA sequence but use a different cutting
site – they are also called isochizomers
• These enzymes cut DNA strands
reproducibly in the same place, which is
extremely useful in gene analysis
4-6
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
4-7
 Restriction-Modification System
• What prevents these
enzymes from cutting up
the host DNA?
– They are paired with
methylases
– Theses enzymes recognize,
methylate the same site
• Together they are called a
restriction-modification
system, R-M system
• Methylation protects DNA,
after replication the parental
strand is already
methylated 4-8
 An 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 4-9
 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
4-10
 Vectors
• Vectors function as DNA carriers to allow
replication of recombinant DNAs
• Typical experiment uses 1 vector plus a
piece of foreign DNA
– Depends on the vector for its replication
– Foreign DNA has no origin of replication, the
site where DNA replication begins
• There are 2 major classes of vectors:
– Plasmids
– Phages 4-11
 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)

4-12
 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
4-13
 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 4-14
 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

4-15
 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
4-16
 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
4-17
 pUC and -galactosidase
Newer pUC plasmids have:
– Ampicillin resistance gene
– Multiple cloning site inserted into the gene
lacZ’ coding for the enzyme -galactosidase
• Clones with foreign DNA in the MCS disrupt the
ability of the cells to make -galactosidase
• Plate on media with a -galactosidase indicator (X-
gal) and clones with intact -galactosidase enzyme
will produce blue colonies
• Colorless colonies should contain the plasmid with
foreign DNA
4-18
 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
4-19
 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 -galactosidase gene
• MCS facilitates directional cloning into 2 different
restriction sites 4-20
 Phages As Vectors
• Bacteriophages are natural vectors that
transduce bacterial DNA from one cell to
another
• Phage vectors infect cells much more
efficiently than plasmids transform cells
• Clones are not colonies of cells using
phage vectors, but rather plaques, a
clearing of the bacterial lawn due to phage
killing the bacteria in that area
4-21
  Phage Vectors
• First phage vectors were constructed by
Fred Blattner and colleagues
– Removed middle region
– Retained genes needed for phage replication
– Could replace removed phage genes with
foreign DNA
• Originally named Charon phage
• More general term, replacement vectors
4-22
 Phage Vector Advantages
• Phage vectors can receive larger amounts
of foreign DNA
– Charon 4 can accept up to 20kb of DNA
– Traditional plasmid vectors take much less
• Phage vectors require a minimum size
foreign DNA piece (12 kb) inserted to
package into a phage particle

4-23
Cloning Using a Phage Vector

4-24
 Genomic Libraries
• A genomic library contains clones of all the
genes from a species genome
• Restriction fragments of a genome can be
packaged into phage using about 16 – 20
kb per fragment
• This fragment size will include the entirety
of most eukaryotic genes
• Once a library is established, it can be
used to search for any gene of interest
4-25
 Plaque Hybridization
• Searching a genomic
library requires probe
showing which clone
contains desired gene
• Ideal probe – labeled
nucleic acid with
sequence matching
the gene of interest

4-26
 Cosmids
Cosmids are designed for cloning large DNA
fragments
– Behave as plasmid and phage
– Contain
• cos sites, cohesive ends of phage DNA that allow the DNA to be
packaged into a  phage head
• Plasmid origin of replication permitting replication as plasmid in
bacteria
– Nearly all  genome removed so there is room for large
inserts (40-50 kb)
– So little phage DNA can’t replicate, but they are
infectious carrying recombinant DNA into bacterial cells
4-27
 M13 Phage Vectors
• Long, thin, filamentous phage M13
• Contains:
– Gene fragment with -galactosidase
– Multiple cloning site like the pUC family
• Advantage
– This phage’s genome is single-stranded DNA
– Fragments cloned into it will be recovered in
single-stranded form

4-28
 M13 Cloning to Recover Single-
stranded DNA Product
• After infecting E. coli cells,
single-stranded phage DNA is
converted to double-stranded
replicative form
• Use the replicative form for
cloning foreign DNA into MCS
• Recombinant DNA infects host
cells resulting in single-stranded
recombinant DNA
• Phage particles, containing
single-stranded phage DNA is
secreted from transformed cells
and can be collected from media
4-29
 Phagemids
Phagemids are also vectors
– Like cosmids have aspects of
both phages and plasmids
– Has a MCS inserted into lacZ’
gene to screen blue staining /
white colonies
– Has origin of replication of
single-stranded phage f1 to
permit recovery of single-
stranded recombinant DNA
– MCS has 2 phage RNA
polymerase promoters, 1 on
each side of MCS
4-30
 Summary
• Two kinds of phage are popular cloning vectors
‑  phage
‑ Has nonessential genes removed making room for
inserts
- Cosmids accept DNA up to 50 kb
- M13 phage
- Has MCS
- Produces single-stranded recombinant DNA
• Plasmids called phagemids also produce single-
stranded DNA in presence of helper phage
• Engineered phage can accommodate inserts up
to 20 kb, useful for building genomic libraries
4-31
 Eukaryotic Vectors and Very
High Capacity Vectors
• There are vectors designed for cloning
genes into eukaryotic cells
• Other vectors are based on the Ti plasmid
to carry genes into plant cells
• Yeast artificial chromosomes (YAC) and
bacterial artificial chromosomes (BAC) are
used for cloning huge pieces of DNA

4-32
Identifying a Specific Clone With
a Specific Probe
• Probes are used to identify a desired clone
from among the thousands of irrelevant
ones
• Two types are widely used
– Polynucleotides also called oligonucleotides
– Antibodies

4-33
 Polynucleotide Probes
Looking for a gene you want, might use
homologous gene from another organism
– If already cloned
– Hope enough sequence similarity to permit
hybridization
– Need to lower stringency of hybridization
conditions to tolerate some mismatches

4-34
 Control of Hybridization
Stringency
• Factors that promote separation of two strands
in a DNA double helix:
– High temperature
– High organic solvent concentration
– Low salt concentration
• Adjust conditions until only perfectly matched
DNA strands form a duplex = high stringency
• Lowering these conditions lowers stringency
until DNA strands with a few mismatches can
hybridize
4-35
 Protein-based Polynucleotide
Probes
No homologous DNA from another organism?
• If amino acid sequence is known, deduce a set of
nucleotide sequences to code for these amino
acids
• Construct these nucleotide sequences chemically
using the synthetic probes
• Why use several?
– Genetic code is degenerate with most amino acids
having more than 1 nucleic acid triplet
– Must construct several different nucleotide
sequences for most amino acids
4-36
 Summary
• Specific clones can be identified using
polynucleotide probes binding to the gene
itself
• Knowing the amino acid sequence of the a
gene product permits design of a set of
oligonucleotides that encode part of the
amino acid sequence
• Can be a very quick and accurate means
of identifying a particular clone
4-37
 cDNA Cloning
• cDNA is the abbreviation for
complementary DNA or copy DNA
• A cDNA library is a set of clones
representing as many as possible of the
mRNAs in a given cell type at a given time
– Such a library can contain tens of thousands
of different clones

4-38
Making a cDNA Library

4-39
 Reverse Transcriptase Primer
• Central to successful cloning is the
synthesis of cDNA from an mRNA
template using reverse transcriptase (RT),
RNA-dependent DNA polymerase
– RT cannot initiate DNA synthesis without a
primer
– Use the poly(A) tail at 3’ end of most
eukaryotic mRNA so that oligo(dT) may serve
as primer
4-40
 Ribonuclease H
• RT with oligo(dT) primer has made a
single-stranded DNA from mRNA
• Need to start to remove the mRNA
• Partially degrade the mRNA using
ribonuclease H (RNase H)
– Enzyme degrades RNA strand of an RNA-
DNA hybrid
– Remaining RNA fragments serve as primers
for “second strand” DNA using nick translation

4-41
 Nick Translation
• The nick translation process
simultaneously:
– Removes DNA ahead of a nick
– Synthesizes DNA behind nick
– Net result moves or translates
the nick in the 5’ to 3’ direction
• Enzyme often used is E. coli
DNA polymerase I
– Has a 5’ to 3’ exonuclease
activity
– Allows enzyme to degrade
DNA ahead of the nick 4-42
 Trailing Terminal Transferase
• Don’t have the sticky ends of genomic DNA
cleaved with restriction enzymes
• Blunt ends will ligate, but inefficient
• Generate sticky ends using terminal
deoxynucleotidyl transferase (TdT), terminal
transferase with one dNTP
– If use dCTP with the enzyme
– dCMPs are added one at a time to 3’ ends of the
cDNA
– Same technique adds oligo(dG) ends to vector
– Generate ligation product ready for transformation 4-43
 Vector Choice
• Choice based on method used to detect
positive clones
• Plasmid or phagemid like pUC or pBS will
be used with colony hybridization and a
labeled DNA probe
• If phage like gt11, cloned cDNA under
control of lac promoter for transcription
and translation of the cloned gene and
antibody screening
4-44
 Rapid Amplification of cDNA
Ends
• If generated cDNA is not full-length,
missing pieces can be filled in using rapid
amplification of cDNA ends (RACE)
• Technique can be used to fill in either the
missing portion at the 5’-end (usual
problem)
• Analogous technique can be used to fill in
a missing 3’-end
4-45
 5’-RACE
• Use RNA prep containing
mRNA of interest and the
partial cDNA
• Anneal mRNA with the
incomplete cDNA
• Reverse transcriptase will
copy rest of the mRNA
• Tail the completed cDNA
with terminal transferase
using oligo(dC)
• Second strand synthesis
primed with oligo(dG)
4-46
 Summary
• Make cDNA library with synthesis of cDNAs one
strand at a time
– Use mRNAs from a cell as templates for 1st strands, then
1st strand as template for 2nd
– Reverse transcriptase generates 1st strand
– DNA polymerase I generates the second strands
• Give cDNAs oligonucleotide tails that base-pair with
complementary tails on a cloning vector
• Use these recombinant DNAs to transform bacteria
• Detect clones with:
– Colony hybridization using labeled probes
– Antibodies if gene product translated
• Incomplete cDNA can be filled in with 5’- or 3’-RACE
4-47
 4.2 The Polymerase Chain
Reaction

• Polymerase chain reaction (PCR) can

yield a DNA fragment for cloning
• PCR is:
– More recently developed
– Very useful for cloning cDNAs

4-48
 Standard PCR
• Invented by Kary Mullis and colleagues in 1980s
• Use enzyme DNA polymerase to copy a selected
region of DNA
– Add short pieces of DNA (primers) that hybridize to DNA
sequences on either side of piece of interest – causes
initiation of DNA synthesis through that area, X
– Copies of both strands of X and original DNA strands are
templates for next round of DNA synthesis
– Selected region DNA now doubles in amount with each
synthesis cycle
• Special heat-stable polymerases able to work after
high temperatures needed to separate strands
make process “set and forget” for many cycles 4-49
Amplifying DNA by PCR

4-50
 Using Reverse Transcriptase
(RT-PCR) in cDNA Cloning
• To clone a cDNA from just one mRNA
whose sequence is known, use type of
PCR called reverse transcriptase PCR
(RT-PCR)
• Difference between PCR and RT-PCR
– Start with an mRNA not double-stranded DNA
– Begin by converting mRNA to DNA
– Next use forward primer to convert ssDNA to
dsDNA
– Now standard PCR continues 4-51
 RT-PCR Can Generate Sticky
Ends
• With care, restriction
enzyme sites can
even be added to the
cDNA of interest
• Able to generate
sticky ends for ligation
into vector of choice
• 2 sticky ends permits
directional cloning

4-52
 Real-Time PCR

• Real-time PCR quantifies the

amplification of the DNA as it occurs
• As DNA strands separate, anneal to
forward and reverse primers, and to
fluorescent-tagged oligonucleotide
complementary to part of one DNA
strand

4-53
 Fluorescent Tags in Real-Time
PCR
• This fluorescent-tagged
oligonucleotide serves
as a reporter probe
– Fluorescent tag at 5’-end
– Fluorescence quenching
tag at 3’-end
• With PCR rounds the 5’
tag is separated from the
3’ tag
• Fluorescence increases
with incorporation into
DNA product
4-54
 4.3 Methods of Expressing
Cloned Genes
Cloning a gene permits
• Production of large quantities of a
particular DNA sequence for detailed
study
• Large quantities of the gene’s product can
also be obtained for further use
– Study
– Commerce

4-55
 Expression Vectors
• Vectors discussed so far are used to first
put a foreign DNA into a bacterium to
replicate and screen
• Expression vectors are those that can
yield protein products of the cloned genes
– For high level expression of a cloned gene
best results often with specialized expression
vectors
– Bacterial vectors have a strong promoter and
a ribosome binding site near ATG codon 4-56
 Fusion Proteins
• Some cloning vectors,
pUC and pBS, can work
as expression vectors
using lac promoter
• If inserted DNA is in the
same reading frame as
interrupted gene, a
fusion protein results
– These have a partial -
galactosidase sequence
at amino end
– Inserted cDNA protein
sequence at carboxyl end 4-57
 Inducible Expression Vectors
• Main function of expression vector is to yield the
product of a gene – usually more is better
• For this reason, expression vectors have very
strong promoters
• Prefer keep a cloned gene repressed until time
to express
– Large quantities of eukaryotic protein in bacteria are
usually toxic
– Can accumulate to levels that interfere with bacterial
growth
– Expressed protein may form insoluble aggregates,
inclusion bodies
4-58
 Controlling the lac Promoter
• lac promoter is somewhat inducible
– Stays off until stimulated
– Actually repression is incomplete or leaky
– Some expression will still occur
• To avoid this problem, express using a
plasmid or phagemid carrying its own lacI
repressor gene, such as pBS

4-59
 Arabinose Promoter
• The hybrid trc promoter combines strength
of the trp (tryptophan operon) promoter
with inducibility of lac promoter
• Promoter from ara operon, PBAD, allow fine
control of transcription
– Inducible by arabinose, a sugar
– Transcription rate varies with arabinose
concentration

4-60
 Tightly Controlled Promoter
• Lambda () phage promoter, PL, is tightly
controlled
• Expression vectors with this promoter-
operator system are used in host cells with
temperature-sensitive  repressor gene
– Repressor functions are low temperatures
– Raise temperature to nonpermissive
temperature, the repressor doesn’t function
and cloned gene is expressed
4-61
 Summary
• Expression vectors are designed to yield
the protein product of a cloned gene
• When a lac inducer is added, cell begins
to make T7 polymerase which transcribes
the gene of interest
• Many molecules of T7 polymerase are
made, so gene is turned on to a very high
level with abundant amount of protein
product made
4-62
 Expression Vectors That
Produce Fusion Proteins
• Most vectors express fusion proteins
– The actual natural product of the gene isn’t made
– Extra amino acids help in purifying the protein product
• Oligohistidine expression vector has a short
sequence just upstream of MCS encoding 6 His
– Oligohistidine has a high affinity for divalent metal ions
like Ni2+
– Permits purification by nickel affinity chromatography
– His tag can be removed using enzyme enterokinase
without damage to the protein product
4-63
Oligohistidine Expression
Vector

4-64
 Fusion Proteins in gt11
• This phage contains
lac control region
and lacZ gene
• Products of gene
correctly inserted
will be fusion
proteins with a -
galactosidase leader

4-65
 Antibody Screening With gt11
• Lambda phages with
cDNA inserts are plated
• Protein released are
blotted onto a support
• Probe with antibody to
protein
• Antibody bound to
protein from plaque is
detected with labeled
protein A
• Partial cDNAs can be
completed with RACE
4-66
 Summary
• Expression vectors frequently produce
fusion proteins
– One part of the protein comes from coding
sequences in the vector
– Other part from sequences in the cloned gene
• Many fusion proteins have advantage of
being simple to isolate by affinity
chromatography
• Vector lgt11 produces fusion proteins that
can be detected in plaques with a specific
antiserum
4-67
 Bacterial Expression System
Shortcomings
• There are problems with expression of
eukaryotic proteins in a bacterial system
– Bacteria may recognize the proteins as
foreign and destroy them
– Posttranslational modifications are different in
bacteria
– Bacterial environment may not permit correct
protein folding
• Very high levels of cloned eukaryotic
proteins can be expressed in useless,
insoluble form 4-68
Eukaryotic Expression Systems
• Avoid bacterial expression problems by
expressing the protein in eukaryotic cell
• Initial cloning done in E. coli using a shuttle
vector, able to replicate in both bacterial and
eukaryotic cells
• Yeast is suited for this purpose
– Rapid growth and ease of culture
– Still a eukaryote with more appropriate
posttranslational modification
– Secretes protein in growth medium so easy
purification 4-69
 Use of Baculovirus As
Expression Vector
• Viruses in this class have a large circular
DNA genome, 130 kb
• Major viral structural protein is made in
huge amounts in infected cells
– Promoter for this protein, polyhedrin, is very
active
– These vectors can produce up to 0.5 g of
protein per liter of medium
– Nonrecombinant viral DNA entering cells
cannot result in infectious virus as it lacks an
essential gene supplied by the vector 4-70
Baculovirus Expression

4-71
 Animal Cell Transfection
• Calcium phosphate
– Mix cells with DNA in a phosphate buffer
– Then solution of calcium salt added to form a
precipitate
– Cells take up the calcium phosphate crystals
which include some DNA
• Liposomes
– DNA mixed with lipid to form liposomes, small
vesicles with some of the DNA inside
– DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell
4-72
 Summary
• Foreign genes can be expressed in
eukaryotic cells
• These eukaryotic systems have advantages
over prokaryotic ones
– Made in eukaryotic cells tend to fold properly
and are then soluble rather than aggregated
into insoluble inclusion bodies
– Posttranslational modifications are made in a
eukaryotic manner
4-73
Using the Ti Plasmid to Transfer
Genes to Plants
• Genes can be introduced into plants with
vectors that can replicate in plant cells
• Common bacterial vector promoters and
replication origins are not recognized by
plant cells
• Plasmids are used containing T-DNA
– T-DNA is derived from a plasmid known as
tumor-inducing (Ti)
– Ti plasmid comes from bacteria that cause
plant tumors called crown galls
4-74
 Ti Plasmid Infection
• Bacterium infects plant, transfers Ti
plasmid to host cells
• T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
• T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
4-75
Ti Plasmid Transfers Crown
Gall

4-76
Use of the T-DNA Plasmid

4-77