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
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.
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 >90˚C,
PCR primers are annealed by
reducing the temperature to ~
50-60˚C, and then the primers
are elongated by a DNA
polymerase. The process is
repeated over many cycles (next
slide).
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 heatresistant 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.
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.
DNA Sequencing (Part 1)
The classical method for DNA
sequencing is dideoxy chaintermination 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.
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.
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.
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.
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.
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.
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).
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.
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).
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 (G418) 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.