Chap. 5 Molecular Genetic Techniques (Part B)

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Chap. 5 Molecular Genetic Techniques (Part B)

• Topics
• DNA Cloning and Characterization (cont.)
• Using Cloned DNA fragments to Study Gene
  Expression

Goals Learn genetic and recombinant DNA methods
for isolating genes and characterizing the
functions of the proteins they encode.
Use of RNA interference (RNAi) in analysis of
planarian regeneration
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Separation of DNA Fragments by Gel Electrophoresis
DNA fragments must be separated and purified
prior to many rDNA procedures. A convenient
method for both separation and purification is
gel electrophoresis. DNA molecules have a uniform
charge to mass ratio. In an electric field they
run towards the anode and are separated based on
size (length) when electrophoresed through a gel
sieving network made of polyacrylamide or agarose
(Fig. 5.19a). DNA bands can be visualized by
radiolabeling the DNA or by noncovalent binding
of the fluorescent dye known as ethidium bromide
(Fig. 5.19b). The region of the gel containing
the band can be excised and the DNA fragment
obtained by extraction with a buffer.
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Polymerase Chain Reaction (Part 1)
The PCR is a method for amplifying a DNA sequence
region located between two primers (Fig. 5.20).
Amplification is specific and highly sensitive,
allowing a target sequence to be specifically
amplified starting from a complex mixture of DNA.
In Cycle 1, double-stranded DNA containing the
target sequence is first denatured by heating to
gt90C, PCR primers are annealed by reducing the
temperature to 50-60C, and then the primers
are elongated by a DNA polymerase. The process is
repeated over many cycles (next slide).
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Polymerase Chain Reaction (Part 2)
In later cycles, most of the DNA synthesized
corresponds exclusively to the sequence region
between the primers. The yield of amplified DNA
increases exponentially based on the cycle
number, n (yield is proportional to 2n). A
heat-resistant DNA polymerase, typically Taq
polymerase from the Yellowstone Archaen organism,
Thermus aquaticus, is used as the DNA polymerase
to prevent its denaturation due to heating.
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Cloning of PCR-amplified DNA
PCR fragments can be readily cloning into a
vector by incorporating restriction enzyme sites
into the ends of the primers used in
amplification (Fig. 5.21). The added sequences do
not interfere with polymerization reactions.
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DNA Sequencing (Part 1)
The classical method for DNA sequencing is
dideoxy chain-termination sequencing (Sanger
sequencing). The basic approach involves 1)
enzymatic synthesis of a set of specifically
labeled (at each base) daughter strands from the
molecule being sequenced that differ by one
nucleotide in length, and 2) separation of the
fragments by electrophoresis. The sequence then
is read from the positions of consecutive
fragments on the gel. Termination at each base is
accomplished using dideoxyribonucleoside
triphosphates (ddNTPs) (figure). These
nucleotides are incorporated into a growing DNA
chain, but block further elongation because they
lack a 3'-hydroxyl group.
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DNA Sequencing (Part 2)
Chain-termination strand synthesis by a DNA
polymerase is illustrated for the G reaction in
the figure at left below. To prevent all chains
from terminating at the first G position, ddGTP
is added at 1/100th the amount of dGTP. To
achieve termination at each type of base, four
separate reactions are run in parallel using the
sequencing template (right, below). Each reaction
is spiked with one of the four ddNTPs.
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DNA Sequencing (Part 3)
The Sanger method of DNA sequencing is being
replaced by so-called next generation sequencing,
which has a greater capacity for sequence
generation. Next generation sequencing currently
is the preferred method for sequencing of entire
genomes. In the method, a large collection of DNA
fragments generated from genomic DNA, for
example, is prepared and attached to a solid
support (Fig. 5.23). PCR is used to amplify each
attached fragment into a cluster of about 1,000
molecules. As many as 3 x 109 discrete clusters
are attached to the final support. Each cluster
derives from a unique fragment in the original
collection.
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DNA Sequencing (Part 4)
The DNA fragments in each cluster then are
sequenced using fluorescently-tagged dNTPs, in
which each species is tagged with a different
color (Fig. 5.24, top). dNTPs are incorporated
one at a time in each cycle of sequencing, and
the color and identity of the dNTP is determined
by fluorescent microscopy (Fig. 5.24, bottom). Up
to 100 cycles of sequencing are performed. Thus,
3 x 1011 total bases of sequence information can
be collected from the 3 x 109 clusters attached
to the support. The sequences of all fragments
are aligned to determine overlapping regions, and
the overlaps are used to assemble the overall
sequence of the starting genomic DNA, for
example.
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Southern Blotting
Southern blotting is a sensitive method for the
detection of a DNA sequence within a mixture
(Fig. 5.26). The DNA first is cleaved with a
restriction enzyme to produce fragments that can
be separated by electrophoresis. After
electrophoresis, DNA fragments are denatured with
alkali and transferred to a nitrocellulose
membrane by capillary action, creating a replica
of the original gel. The membrane is incubated
with a labeled probe which binds to and detects
the fragment of interest.
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Northern Blotting
Northern blotting is a method for the detection
of a specific mRNA in a mixture of RNAs. Like
Southern blotting the method is highly specific
and sensitive. RNA from a cell/tissue is
extracted and separated by electrophoresis. As in
Southern blotting the RNA is transferred to a
nitrocellulose membrane and incubated with a
labeled probe that is complementary to the RNA.
As shown in Fig. 5.27, the method can be use to
quantitate transcription of a mRNA under
different cellular conditions.
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DNA Microarrays and Transcriptome Analysis
A complete analysis of all the mRNAs transcribed
in an organism (transcriptome) can be performed
by DNA microarray analysis (Fig. 5.29). In this
method, mRNA is isolated and converted to cDNA,
and then labeled with a fluorescent dye. The cDNA
is hybridized to a gene chip containing
oligonucleotide sequences representing all or a
subset of genes in the organism. The amount of
mRNA expressed from each gene is determined by
quantitation of fluorescence intensity of the
cDNA bound to each probe. The method can be
adapted to compare gene expression levels in
cells under different growth conditions, etc.
(Fig. 5.29).
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Cluster Analysis of Gene Expression
DNA microarray data can be analyzed to identify
clusters of genes with related functions that are
similarly regulated under certain conditions
(Fig. 5.30). As an illustration, clusters of
coordinately regulated fibroblast genes that
switch on or off in response to a change in media
can be identified by analyzing gene expression
data from several microarray experiments
conducted over time. The broad functions of the
clusters can be assigned (e.g., cell cycle
control) based on the functions of known genes
within the cluster. Cluster analysis therefore is
a powerful method for deducing the functions of
unknown genes.
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Expression of Recombinant Proteins in E.coli
Eukaryotic and prokaryotic proteins commonly are
synthesized in E. coli for pharmaceutical and
research applications. All that is required is to
clone the gene of interest under the control of a
strong E. coli promoter in a plasmid expression
vector (Fig. 5.31). Promoters such as the lac
promoter permit inducible expression by lactose,
or more commonly, the synthetic lactose analog
isopropylthiogalactoside (IPTG). Bacterial genes
can be directly expressed in E. coli because they
lack introns. cDNAs (which also lack introns) are
used for expression of eukaryotic proteins in E.
coli. A tag (e.g., His6) can be added to the N-
or C-terminus of the protein to expedite
purification (by nickel affinity chromatography).
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Expression of Cloned Genes in Cultured Animal
Cells
Plasmids carrying cloned eukaryotic genes can be
introduced into animal cells grown in culture by
transfection. In transient transfection (Fig.
5.32a), the introduced plasmid contains a viral
replication origin, which allows it to propagate
for a short time until diluted out in the cells
due to inefficient segregation. In stable
transfection (transformation) (Fig. 5.32b), the
plasmid lacks a replication origin. Thus, a
selection based on the neor marker (G-418) is
carried out to obtain cells in which the plasmid
has integrated into the genome. Expressed
proteins, which can be glycosylated, etc., are
purified from transfected cells for analysis.