Transcript chap-4 - Workforce3One

Molecular Cloning Methods
Chapter 4
Vectors
• Vectors - 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
• There are 2 major classes of vectors:
– Plasmids
– Phages
pBR322 Plasmid
• pBR322 - cloning
methods
• Resistance for 2
antibiotics
• Tetracycline
• Ampicillin
– Origin of replication
between the 2 resistance
genes
– Only 1 site for several
restriction enzymes
Plasmids As Vectors
• 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)
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
Bacterial Transformation
• Traditional method - incubating bacterial cells
in concentrated calcium salt solution
– The solution makes the cell membrane permeable to the plasmid DNA
• Newer method - high voltage to drive the DNA
into the cells - electroporation
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
Screening With Replica Plating
• Replica plating transfers clone
copies from original
tetracycline plate to a plate
containing ampicillin
• 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
• 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 colonies should contain the plasmid with
foreign DNA
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
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 – plaques - a clearing of the bacterial
lawn due to phage killing the bacteria in that
area
Phage Vectors
• First phage vectors were constructed by Fred
Blattner and colleagues
– Removed middle region
– Replaced with foreign DNA
– Retained genes needed for phage replication
• Charon phage
• General term - replacement vectors
Cloning Using a Phage Vector
• Phage vectors can
receive larger amounts
of foreign 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
Genomic Libraries
• A genomic library contains clones of all the
genes from a genome
• Restriction fragments of a genome can be
packaged into phage using about 16 – 20 kb
per fragment
• Once a library is established - it can be used to
search for any gene of interest
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
Homework
• Explain Cosmids, M13 phage vectors and
Phagemids and Eukaryotic vectors and
very high capacity vectors– their features
and functions
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
Polynucleotide Probes
• Use homologous gene from another
organism
– If already cloned
– Enough sequence similarity to permit
hybridization
– Need to lower stringency of hybridization
conditions to tolerate some mismatches
Control of Hybridization
Stringency
• 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
Protein-based Polynucleotide
Probes
• 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
cDNA cloning
• A cDNA library is a set of clones
representing many mRNAs in a given cell
type at a given time
– Such a library can contain tens of thousands
of different clones
– One cDNA – a clone containing a DNA copy
of just one mRNA
Making a cDNA library
Role of RT in making a cDNA library
• Synthesis of cDNA from an mRNA
template using reverse transcriptase
(RT)
– 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
Role of Rnase H in making a cDNA
library
• Partially degrade the mRNA using
ribonuclease H (RNase H)
– Enzyme degrades RNA strand of an RNADNA hybrid
– Remaining RNA fragments serve as
primers for “second strand” DNA using
nick translation
Nick translation
– DNA polymerase I
shows 5’ to 3’
exonuclease activity
– Removes/degrades
DNA ahead of a nick
– Synthesizes DNA
behind nick
– Net result moves or
translates the nick in
the 5’ to 3’ direction
Role of terminal transferase in making
a cDNA library
• Generate sticky ends using terminal
deoxynucleotidyl transferase (TdT), terminal
transferase with one dNTP
– 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
How to detect positive clones?
• Plasmid or phagemid like pUC or pBS
will be used with colony hybridization and
a labeled DNA probe
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) or fill in a missing 3’-end
5’-RACE procedure
• 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)
Polymerase Chain Reaction
• Polymerase chain reaction (PCR) can
yield a DNA fragment for cloning
• Invented by Kary Mullis and colleagues in
1980s
• PCR is:
– More recently developed
– Very useful for cloning cDNAs
PCR
Reverse Transcriptase PCR (RT-PCR)
in cDNA cloning
• To clone a cDNA from
just one mRNA
whose sequence is
known - reverse
transcriptase PCR
(RT-PCR)
Reverse Transcriptase PCR (RT-PCR)
in cDNA cloning
• 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
RT-PCR Generates Sticky Ends
• Restriction enzyme
sites can be added to
the cDNA of interest
• Able to generate
sticky ends for
ligation into vector
of choice
• 2 sticky ends permits
directional cloning
Fluorescent Tags in Real-Time
PCR
• 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
Methods of Expressing Cloned
Genes
• Expression vectors are those that can
yield protein products of the cloned
genes
– For high level expression of a cloned gene
– Bacterial vectors have a strong promoter and
a ribosome binding site near ATG codon
Inducible expression vectors
• 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
Expression vectors produce
fusion proteins
Expression vectors 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
Bacterial Expression System Shortcomings
– Problems - 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
How to avoid bacterial expression
problems?
• Initial cloning done in E. coli using a
shuttle vector - replicate in both bacterial
and eukaryotic cells
• Yeast is suited for this purpose
– Rapid growth and ease of culture
– Eukaryote with more appropriate
posttranslational modification
– Secretes protein in growth medium - easy
purification
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 polyhedrin protein is very active
– These vectors can produce up to 0.5 g of
protein per liter of medium
Use of Baculovirus As Expression
Vector
Formation of crown gall tumors
Agrobacterium tumefaciens
• Bacterium infects plant - transfers Ti
plasmid to host cells
• T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells – galls/tumors
• 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
Using Ti-plasmid to transfer genes to
plants
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