MBB432: Advanced Molecular Biology Techniques

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MBB432 Advanced Molecular Biology
Techniques -traditional genetics involves
working towards the gene from a mutant phenotype,
but today one is more often taking a reverse
genetics approach in which there is the desire
to study a cloned, sequenced gene -in the era of
genomics there is an even greater need than
before for tools that can allow one to rapidly
and effectively characterize gene function -in
the lectures and labs of this course we will
explore some of the advanced molecular techniques
being used to study and manipulate
genes Outline of topics to be covered 1.
Targeted alteration of gene function through use
of oligonucleotides Lecture Lab a.
Site-directed mutagenesis techniques a.
Mutagenesis of Drac1 gene b. Gene
silencing with RNAi N/A c. Prevention of
translation by morpholinos N/A 2. Expressing
proteins from cloned genes for biochemical
studies/antibody production Lecture Lab a.
Expression in bacteria using various fusion a.
Expression of GST fusion protein protein
approaches of Drac1
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2. Expressing proteins from cloned genes
continued Lecture Lab b. Expression
using baculovirus vectors in N/A insect
cells c. Expression using vectors in yeast
cells N/A 3. Transgenic animal approaches
Lecture Lab a. Use of transgenes to study
gene function a. Use of GAL4-UAS system in
non-vertebrate model organisms in
Drosophila b. Use of transgenes to study gene
function b. N/A in mice 4. Gene expression in
mammalian cells- lecture only 5. Two-hybrid
approaches for looking at protein-protein
interactions Lecture Lab a. Yeast
two-hybrid approaches a. Screening for binding
partners for the Drac1 effector prote
in POSH using yeast two-hybrid
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5. Two-hybrid approaches for looking at
protein-protein interactions continued Lecture
Lab b. Bacterial two-hybrid
approaches N/A c. Mammalian two-hybrid
approach N/A 6. Looking at mRNA and proteins in
cells/whole organisms Lecture Lab a. RNA
in situ hybridization a. Looking at Drac1
expression in embryos using RNA in
situ hybridization b. Examining RNA
localization in live N/A tissues c. Use of
GFP to visualize proteins in live c. Demo of
GFP in live tissues Drosophila embryos d.
Use of FRET to visualize protein-protein
N/A interactions in live cells
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1. Targeted alteration of gene function through
use of oligonucleotides
a. Site-directed mutagenesis techniques
-site-directed mutagenesis was pioneered by the
late Michael Smith of UBC, who shared the 1993
Nobel Prize in Chemistry with Kary Mullis
-Smith had been working on techniques for
synthesizing oligonucleotides when he realized
that such oligonucleotides could be used to
introduce specific mutations in a DNA
sequence -he tested his idea using the phage
fX174, which, like M13, is a single-stranded
circular DNA phage -Smith synthesized an
oligonucleotide that was complementary to a short
stretch of phage sequence except for a single
base change -the second strand was synthesized
with DNA polymerase using the oligonucleotide as
a primer to produce the double-strand RF form
which was introduced into E. coli -mutant
progeny phage with the single base pair change
were isolated from the bacteria at a frequency of
about 15

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Some uses for site-directed mutagenesis

1) Study of protein
structure-function relationships -catalytic
mechanisms -protein folding -defining the
interactions of proteins with other molecules
such as ligands, substrates, receptors, partners
in signaling pathways 2) Reverse genetics -the
classical genetic approach to studying gene
function is to start with a mutant phenotype and
work towards identifying the gene/protein whose
defect is causing that mutant phenotype -now,
more and more genes are first identified as
cloned sequences, and are being studied through
reverse genetics, where one starts with the gene
and works towards a mutant phenotype -a common
approach in reverse genetics is to introduce
mutations into a cloned gene through
site-directed mutagenesis and place the mutated
transgene into cells or organisms -in the lab we
will see how such an approach can be taken
through experiments using the Drac1 gene of
Drosophila
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-in the 25 years since Smith first described
site-directed mutagenesis, a variety of
approaches have been developed which maximize
the proportion of mutants recovered (to much
higher than Smiths 15) -many
commercially-available kits are on the market
which promise up to 100 mutants by having a
selection step that favours a mutated
plasmid -in the lab, we will be using the
QuikChange site-directed mutagenesis kit from
Stratagene -the desired mutation is introduced
into the target sequnece (residing in a plasmid
vector such as Bluescript) using a pair of
oligonucleotides with the appropriate base pair
changes -the primers are reverse complements of
each other, so both strands of the target plasmid
will be mutated -as an example, we can consider
the control mutagenesis reaction supplied with
the QuikChange kit -for this control, the
defective b-galactosidase gene of the
pWhitescript plasmid, which contains a stop codon
(TAA) where a glutamine codon (CAA) would
normally reside, is repaired using the pair of
mutagenic oligonuclotides shown below
Base change converting T back to C 5 CCA TGA
TTA CGC CAA GCG CGC AAT TAA CCC TCA C 3 5
GTG AGG GTT AAT TGC GCG CTT GGC GTA ATC ATG G 3

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-as shown in the figure, mutated sequences in
the QuikChange protocol are selected for based on
their lack of methylation -recall that in E.
coli cells (where the target plasmid is grown up)
DNA Sequences are methylated by methylases -the
dam methylase in E. coli methylates adenine in
the sequence 5GATC 3 -the mutated DNA
synthesized in vitro will not be methylated -
the unmutated, parental DNA can be digested
with the restriction enzyme Dpn I (from
Diplococcus pneumoniae ), which cuts DNA
methylated on adenine in the sequence 5GATC
3 -this leaves the two mutated strands
available for replication in the XL1-Blue strain
of E. coli
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-other site-directed mutagenesis systems use
additional mutagenic primers (in addition to
that mutating the target sequences) to alter the
plasmid vector in such a way that it can be
selected for - in the Altered Sites kit
marketed by Promega, mutant plasmids are
selected on the basis of antibiotic
resistance -the sequence to be mutated must be
subcloned into the pALTER vector, which is
sensitive to ampicillin and resistant to
chloramphenicol -during the mutagenesis
reaction, 3 different oligos are annealed to one
strand of the target plasmid 1. The oligo that
will mutate the insert 2. An oligo mutating the
Amp gene to resistance 3. An oligo mutating the
Cm gene to sensitivity -the mutant strand is
synthesized in vitro, forming a double- stranded
plasmid with base pair mismatches -the hybrid
mutated/parental plasmid is tranformed into a
mutS strain of E. coli, which is incapable of
carrying out mismatch repair -bacteria are
grown on ampicillin, which will select for
plasmids replicated from the mutant
strand -additional rounds of mutagenesis can be
carried out by cycling between antibiotic
resistances
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-in the Transformer kit from Clontech,
selection is carried out by mutating a
unique restriction site in the parental
plasmid -the in vitro synthesized hybrid
mutated/ parental plasmid reaction is cut with
the selection restriction enzyme, this
will Remove intact parental plasmid -the hybrid
mutated/parental plasmid is transformed into
mutS cells, and plasmid DNA then prepared from
these cells -this DNA is then digested with the
selection Enzyme, which will cut plasmid
replicated From the parental strand of the hybrid
plasmid -DNA is then re-transformed into E.
coli, from which mutated plasmid DNA can be
obtained at high frequency -by mutating the
unique restriction site to another unique
restriction site in each round of mutagenesis,
multiple rounds of mutagenesis can be performed
on plasmid
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-site-directed mutagenesis can also be carried
out efficiently using PCR - it is possible to
perform PCR with oligonucleotide primers that
imperfectly match the template sequence, a
feature that obviously lends itself to
site-directed mutagenesis -in theory, PCR
mutagenesis should produce virtually 100 mutants
as the original wild-type template DNA will form
a tiny proportion of total DNA after
amplification -in the simplest scheme, one can
envisage doing a normal PCR, except that one of
the primers is mutagenic lt-----------
primer 2 gene -----------------------------------
--------------------------------
--------------------------------------------------
----------------- ----------gt primer 1
(mutagenic) result is mutagenized gene
fragment ---------------------------------
--------------- -----------------------------
------------------- Problem How to attach the
mutagenized fragment to the rest of the
gene?
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-need a unique restriction site flanking the
mutation (arrow) in the amplified fragment that
is not found elsewhere in gene or cloning
vector-highly unlikely! Solution Mutagenesis by
overlap extension -in this technique, the gene
is separately amplified in two overlapping
fragments, with the mutation in the region of
overlap primer B primer D
lt------- lt-------
--------------------------------------------------
------------- --------------------------------
------------------------------- --------gt
-------gt primer A primer C
PCR 1 PCR 2 -two separate
reactions ------------------------------- ---
---------------------------- product
AB -------------------------------------
-------------------------------------
product CD
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-these overlapping, intermediate products are
then mixed, melted and reannealed -the top
strand of AB can anneal to the bottom strand of
CD, such that the two strands act as primers on
one another in the synthesis of the mutated gene
as the product AD ------------------------
---gt.gt ltlt---------------------
---------------- ------------------------
------------------------------
-------------------------------------------------
----- Targeted alteration of gene function
through use of oligonucleotides continued b.
Gene silencing with RNAi -for years researchers
have been using antisense RNA or DNA as a means
to inhibit the function of target
genes -antisense RNA or DNA forms a hybrid with
the endogenous mRNA transcript and may interfere
with its function in a number of ways, including
the prevention of translation of the mRNA into
protein, or, in the case of antisense DNA,
targeting the mRNA for degradation by RNAse
H -problems with antisense approaches include
specificity, reliability and toxicity -several
years ago a more effective means of inhibiting
gene function with oligonucleotides was
discovered RNAi
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-the efficacy of RNAi was first demonstrated in
C. elegans, but it has since been to find to work
in a diverse range of organisms including
Drosophila and mice -RNAi involves the silencing
of a gene by introduction of double-stranded RNA
(dsRNA) corresponding to sequences within the
gene -to make dsRNA, one needs to transcribe
both sense and antisense strands of RNA from a
cDNA and allow them to anneal -various tricks
have been used to accomplish this -one approach
is to make PCR primers against the target cDNA
which have T7 RNA polymerase binding sites
hanging off the 5 ends -following PCR, we will
have the cDNA sequence with T7 RNA polymerase
binding sites at both ends

cDNA
T7 site
PCR
T7 site
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-following PCR, RNA is transcribed using T7 RNA
polymerase and the two RNA products
annealed -another approach is to clone the cDNA
of interest into a modified pBluescript vector
with a T7 polymerase binding site on both sides
of the insert -once dsRNA has been produced it
can be introduced into the cell or organsim by
various means, including injection -the
mechanism appears to utilize a gene silencing
system present in the organism and a models for
RNAi function are being developed -upon
introduction, the dsRNA becomes bound by a
nuclease that degrades the dsRNA into small
21-25 nucleotide fragments (siRNA) that remain
bound to the enzyme -this siRNA then allows
sequence-specific association of the nuclease
with the target mRNA -RNA helicase activity
promotes strand exchange, such that the sense
strand of the siRNA is replaced with the
mRNA -the mRNA is then cleaved into fragments of
21-25 nucleotides, thus preventing translation
into protein -an intriguing feature of RNAi is
its persistence for long periods of time after
introduction of the dsRNA trigger -the
inactivation of the target mRNA can persist
through cell division, spread to untreated cells,
and, in C. elegans, can even be inherited by
subsequent generations -one component of the
persistence is likely to be re-use of the
nuclease in multiple rounds of mRNA cleavage
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-recent studies indicate that an RNA-directed RNA
polymerase (RdRP) carries out production Of fresh
dsRNA trigger, thus amplifying the original RNAi
effect -this occurs through an siRNA-primed RdRP
reaction that converts target mRNA into dsRNA, as
well as possibly replicating trigger dsRNA
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- a novel approach being taken by various groups
working on Drosophila and C. elegans is
to express dsRNA from an integrated
transgene -the dsRNA transgene is made by
joining a cDNA sequence to an inverted repeat
copy of itself, with a heterologous spacer
between the inverted repeats -when the transgene
is transcribed, it will snap into a hairpin RNA
which can function as a dsRNA trigger for
RNAi -the transgene can be expressed from an
inducible promoter, for example it can be
expressed in different tissues using the
GAL4-UAS system
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Targeted alteration of gene function through use
of oligonucleotides continued c. Prevention of
translation by morpholinos -morpholino oligos
are DNA analogs that appear to be effective
blockers of translation when they are
complementary to the 5 leader sequence of a
target mRNA -they differ from DNA in that the
ribose sugar moiety has been converted to a
morpholine moiety and they use a phosphorodiamidat
e backbone with neutral charge rather than the
negatively charged phosphodiester backbone -they
display little toxicity and have no known
cellular binding proteins or nucleases -studies
to date indicate that they have very specific
effects and work in zebrafish indicates that they
can effectively phenocopy loss-of-function
mutations in genes -a collection of 17 zebrafish
genes with known loss-of- function phenotypes was
targeted with morpholinos, with 16/17 showing
good phenocopies -though most extensively used
in zebrafish, morpholinos have been used in a
variety of organisms including sea urchin,
Xenopus, chick, and mouse