BCHOBI 812 Biochemistry for Dental Students

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Chapter 6 Exploring Genes

• Recombinant DNA technology has revolutionized
  biochemistry and molecular biology
• Basic tools of gene exploration include
• Restriction enzyme analysis
• Blotting techniques
• DNA sequencing
• Solid-phase synthesis of nucleic acids
• The polymerase chain reaction (PCR)

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Restriction Enzymes
Restriction enzymes (restriction endonucleases)
split DNA into specific fragments These enzymes
recognize specific base sequences in DNA and
cleave both strands at specific
points Restriction enzymes are found in
prokaryotes - their normal job is to cleave
foreign DNA The prokaryotic DNA is not degraded
because cleavage sites are protected via
methylation
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(No Transcript)
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Restriction Enzymes
Over 100 restriction enzymes have been identified
and purified They are named after the organism
they come from (e.g. Eco E.coli ) DNA molecules
can be cleaved into smaller, more manageable
parts using restriction enzymes
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Restriction Fragments
Fragments of DNA cleaved by restriction enzymes
are restriction fragments These can be separated
using gel electrophoresis Gels of differing
porosity can be used to separate fragments
ranging from 1,000 base pairs in size, up to
20kb 1kb 1,000 base pairs Resolution can be
as good as separating fragments that differ by a
single base pair
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Southern Blotting

• Restriction fragments containing a specific
  sequence can be identified using a complementary
  DNA strand that has been labeled (e.g. with 32P)
• Mixture of restriction fragments is separated by
  electrophoresis and denatured to form single
  strands
• Fragments are transferred (blotted) onto a
  nitrocellulose sheet
• A DNA probe labeled with 32P is added - this
  hybridizes with complementary fragments
• Autoradiography then reveals the positions of
  fragments complementary to labeled probe

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Southern Blotting
This technique can also be applied to RNA -
Northern blotting
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Blotting
Southern (or DNA) blotting - named after Edwin
Southern, used to identify specific DNA
fragments Northern (or RNA) blotting - used to
identify specific RNA fragments Western (or
protein) blotting - used to identify specific
proteins using antibodies
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DNA Sequencing via the Sanger Dideoxy Method
Fragments of DNA are generated through controlled
interruption of enzymatic replication Same
procedures used on four reaction mixtures at the
same time DNA polymerase is used to copy a
particular sequence of single-stranded DNA A
primer obtained from restriction enzyme digest or
through synthesis is used
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DNA Sequencing
In addition to DNA polymerase, a primer plus the
DNA of interest, radioactively labeled
deoxyribonucleoside triphosphates are used Each
of the four incubation mixtures also contains a
small amount of a 2,3-dideoxy analog of one of
the four deoxyribonucleoside triphosphates Each
time a 2,3-dideoxy analog is incorporated into
the primer chain, replication stops since the
analog lacks a 3-hydroxyl group This generates
fragments with a known 3-terminus
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DNA Sequencing
Mixtures of fragments can be electrophoresed and
DNA sequence read from autoradiogram
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DNA Sequencing
Fluorescent labels can be used instead of
radiolabels (32P) Fluorescent tags are attached
to the primers - a different color tag for each
of the four reaction sets (one for each
base) Fluorescence color will then correspond to
3-terminal base type
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DNA Sequencing
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DNA Sequencing
Whole genomes can be sequenced Sanger and
co-workers sequenced the first, the fX174 DNA
virus, in 1977 Many viruses and bacteria, plus
eukaryotes have had their genomes sequenced The
human genome is (almost) completely sequenced
Diagram of Haemophilus influenzae genome
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DNA Synthesis
Synthesis uses solid-phase methods (growing chain
is linked to an insoluble support) Activated
monomers are deoxyribonucleoside
3-phosphoamidites (blocked nucleotides) Step 1
3-phosphorous of activated monomer is attached
to 5-OH of chain to form a phosphite
triester 5-OH of activated monomer is unreactive
because it is blocked by a dimethoxytrityl (DMT)
group and 3 phosphoryl group is blocked by
b-cyanoethyl (bCE)
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DNA Synthesis
Step 2 Phosphite triester is oxidised by iodine
(I2) to form a phosphotriester Step 3 DMT group
is cleaved by dichloroacetic acid DNA is now
elongated by one unit and is ready for next
cycle At end of synthesis, chain is cleaved from
insoluble support, and is purified by
high-performance liquid chromatography (HPLC)
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DNA Synthesis
DNA chains up to 100 nucleotides long can be
synthesized
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Polymerase Chain Reaction (PCR)
PCR is a method for amplifying specific DNA
sequences Consider a DNA duplex consisting of a
target sequence surrounded by nontarget DNA The
target sequence can be amplified (millions of
copies made) if the flanking sequences are known
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Polymerase Chain Reaction (PCR)
A PCR cycle has three steps A) Strand
separation. The two strands of the parent DNA
are separated by heating to 95C for
15s. B) Hybridization of primers. Solution is
abruptly cooled to 54C to allow each primer to
hybridize to a DNA strand. Primers are present
in large excess, preventing the parent duplex
from reforming. C) DNA synthesis. Solution is
heated to 72C, the optimal temperature for Taq
DNA polymerase (from Thermus aquaticus).
Polymerization is allowed for 30s, synthesizing
copies of both strands.
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Polymerase Chain Reaction (PCR)
The PCR cycle can be repeated many times just by
changing the temperature of the solution - all
components can remain in the one mixture All new
DNA strands can act as templates in successive
cycles After n-cycles, the target sequence is
amplified 2n-fold
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Polymerase Chain Reaction (PCR)
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PCR is a Powerful Diagnostic Tool
Used in medicine Can readily detect bacteria and
viruses using PCR - can find very tiny amounts
before infection is evident Used in
forensics DNA fingerprinting
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Recombinant DNA
Genes can be cloned - amplified many times over -
by introducing them into various cell lines where
they are replicated e.g. a human gene in
E.coli This is achieved by attaching (splicing) a
DNA fragment of interest to a DNA vector A vector
is a piece of DNA that can replicate autonomously
in the appropriate host Restriction enzymes and
DNA ligase are the key tools
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Recombinant DNA
A vector is prepared for splicing by cleaving at
a specific site using a restriction enzyme The
staggered cuts made by a restriction enzyme
produce complementary single-stranded ends
(cohesive or sticky ends) Any DNA fragment can be
inserted at the splice site if it has the same
cohesive ends The DNA fragment and cut vector can
be joined by an enzyme called DNA ligase DNA
ligase catalyzes the formation of a
phosphodiester bond at a break in a DNA chain
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Recombinant DNA
Some vectors will anneal to themselves rather
than to the DNA fragment, leading to inactive
vectors
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Plasmids and Phage Choice Vectors
Plasmids and phages are the vectors of choice for
cloning in bacteria (e.g. E.coli) Plasmids are
circular duplex DNA molecules that occur
naturally in some bacteria These are accessory
chromosomes that can replicate independently of
the host chromosome Plasmids can be (and have
been) modified to carry various useful genes and
have restriction sites added
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Plasmids pBR322
pBR322 is a particularly useful plasmid It
carries genes for resistance to tetracycline and
ampicillin (an antibiotic) It has several
restriction sites Using restriction enzymes and
DNA ligase, genes can be inserted at any of these
sites Insertion of genes at the HindIII, BamHI or
SalI sites inactivates the tetracycline
resistance gene
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Plasmids pBR322
Cells containing pBR322 with the tetracycline
resistance gene inactivated are resistant to
ampicillin, but sensitive to tetracycline,
leading to easy selection Cells that fail to take
up an active vector remain sensitive to both
tetracycline and ampicillin
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l Phage
l phage is another useful vector This is a
bacteriophage that can destroy its host, or can
become part of it In the lytic pathway, viral
genes are fully expressed, eventually leading to
host death In the lysogenic pathway, phage DNA
becomes inserted into the host genome and can be
replicated along with the host DNA Environmental
factors control which pathway is taken
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l Phage
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l Phage
Mutant l phages have been constructed for cloning
purposes One useful form is lgt-lb, which
contains only two EcoRI sites rather than the
normal five This allows the middle 25 of the DNA
to be removed and a suitably long piece
inserted The altered phage can then be used to
infect bacteria for cloning purposes
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l Phage
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Cloning from Digests
Specific genes can be cloned from a digest of
genomic DNA A sample of DNA is sheared, either
mechanically or using restriction enzymes, into
large pieces This is a nearly random process that
results in a population of overlapping DNA
fragments which are separated via gel
electrophoresis Pieces 15kb long are isolated
and synthetic linkers are added to the
ends Cohesive ends are formed from the linkers
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Cloning from Digests
Fragments with cohesive ends are inserted into a
phage vector E.coli are infected with the
vector The resulting lysate contains fragments of
human DNA housed in a sufficiently large number
of virus particles that nearly the entire genome
is represented This is a genomic library
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Cloning from Digests
Phage can be propagated indefinitely, allowing
the genomic library to be used repeatedly Genomic
libraries are screened for the gene of
interest This is done via DNA hybridization Recom
binant phage are placed on a lawn of
bacteria Where phage particles infect the
bacteria a plaque containing identical phage
develops
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Cloning from Digests
A replica of the plate is made by plating onto a
nitrocellulose sheet This is treated with NaOH,
which lyses intact bacteria and denatures DNA A
labeled probe (DNA) specific for the gene is
added and will hybridize with complementary DNA
Presence of the gene is then detected using an
autoradiogram
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Chromosome Walking
The development of yeast artificial chromosomes
(YACs) allows for cloning of very large
genes YACs contain An autonomous replication
sequence (ARS, where replication begins) Two
telomeres (normal ends of eukaryotic
chromosomes) Selectable marker genes A cloning
site
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Chromosome Walking
Large fragments (450kb) of genomic DNA are
separated and ligated in YACs These are then
efficiently replicated in yeast The fragments
ligated into YACs are produced by random cleavage
and so some overlap one another in sequence If a
probe, A, for gene A (perhaps one of the marker
genes) is hybridized with a YAC fragment
containing A, the fragment may also contain a
gene B B can be cleaved and subcloned and a probe
prepared...
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Chromosome Walking
The B probe can then be used to isolate all
occurrences of B in the library, some of which
may contain C In this way, one can walk down
the chromosome
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Complementary DNA (cDNA)
Can E.coli (or other bacteria) be used to clone
and express human DNA ? Human genes have exons
and introns, bacteria do not (No
spliceosome) This limitation can be overcome by
using recombinant DNA that is complementary to
mature (spliced) mRNA The key to doing this is
the enzyme reverse transcriptase
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Complementary DNA (cDNA)
Reverse transcriptase is used by some viruses
(e.g. HIV-1) to form DNA-RNA hybrids as the first
step in replicating their genomes Reverse
transcriptase will synthesize DNA complementary
to an RNA template when given a primer
base-paired to the RNA Most eukaryotic mRNAs have
a poly(A) sequence at their 3 ends A poly(T)
primer can then be used with reverse
transcriptase to synthesize a complementary DNA
(cDNA)
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Complementary DNA (cDNA)
When RNA-cDNA hybrid has been formed, the RNA can
be hydrolyzed by raising the pH (DNA is resistant
to alkalis) The complementary strand can then be
synthesized by adding a new primer The enzyme
terminal transferase adds several nucleotides,
say dGs, to the 3 end - oligo(dC) can then be
used as a primer Synthetic linkers can then be
added, cohesive ends formed and the cDNA can be
inserted into a vector
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Complementary DNA (cDNA)
Using mature mRNA means than the introns are not
present and a human gene can be expressed by
E.coli
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Gene Expression
Most genes are present in the same quantities in
every cell (one copy in haploid cells and two in
diploid cells) The level of expression varies
widely between different cell types (as indicated
by mRNA quantities) Gene expression levels vary
as a function of cell type and as cells respond
to changes in physiological circumstances
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Gene Expression Gene Chips
Gene expression can be analyzed using DNA
microarrays or chips These are constructed by
fixing oligonucleotides to a solid support Each
oligonucleotide is complementary to part of a
specific gene, and its position on the support is
known Fluorescently labeled cDNA is hybridized to
the chip Differences in fluorescence levels
reveals the differences in expression level of
each gene
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Gene Expression Gene Chips
Gene expression in 84 breast tumors
Gene expression in yeast under differing
conditions
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New Genes Inserted in Eukaryotic Cells
Bacteria are ideals hosts for the amplification
of DNA molecules They can also produce large
amounts of prokaryotic and eukaryotic
proteins However, many eukaryotic proteins are
posttranslationally modified - bacteria cannot do
this since they lack the correct enzymes Many
eukaryotic genes are best expressed in eukaryotic
systems
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New Genes Inserted in Eukaryotic Cells
Recombinant DNA can be introduced into animal
cells in several ways 1) Foreign DNA precipitated
by calcium phosphate are taken up by animal
cells A small fraction of this DNA will be
incorporated into the cells genome - the
efficiency is low 2) DNA can be microinjected
into cells A fine-tipped glass micropipette is
inserted into the nucleus and a DNA solution is
injected. About 2 of injected cells are viable
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New Genes Inserted inEukaryotic Cells
3) Use of viral vectors Most effective are
retroviruses which do not usually kill host cells
Engineered viral vectors can be used to infect
eukaryotic cells, with the engineered genes being
incorporated into the host genome with high
efficiency
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Transgenic Animals
By introducing foreign genes on recombinant DNA
into a fertilized egg, a transgenic animal can be
created For example, giant mice can be created
by introducing many copies of a gene for a growth
hormone
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Gene Disruption
The function of a gene can be probed by
inactivating it and looking for the consequences
(gene disruption or knock-out) These methods rely
on homologous recombination - regions of DNA with
strong sequence similarity can exchange
segments If the sequence of a gene is known, in
inactive form can be synthesized and inserted
into a cell, inactivating the gene
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Gene Disruption
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Tumor-Inducing Plasmids and Plant Cells
A common soil bacterium, Agrobacterium
tumefaciens infects and introduces foreign genes
into plants A lump of tumor tissue grows at the
site of infection (crown galls) Crown galls
synthesize opines, a group of amino acid
derivatives, that are metabolized by the
infecting bacteria Tumor-inducing (Ti) plasmids
from this bacterium can be used to introduce new
genes into plant cells
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Tumor-Inducing Plasmids and Plant Cells
The instructions for opine synthesis and tumor
formation are located on a plasmid Part of this
plasmid in incorporated into the plant genome
Ti plasmid derivatives can be used as vectors to
introduce new genes into many, but not all,
plants, creating a genetically modified organism
(GMO)
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Site-Specific Mutagenesis
Recombinant DNA makes it possible to introduce
changes in amino acids into proteins Insertions,
deletions, substitutions and designer genes A
deletion can be introduced by cleaving a plasmid
at two sites and religating to form a smaller
circle This usually removes a fairly large block
of DNA Smaller deletions are created by cleaving
at a single site and then removing single bases
using an exonuclease, then religating
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Site-Specific Mutagenesis
Single amino acid substitutions can be made by
oligonucleotide-directed mutagenesis A particular
substitution can be made if 1) you have a
plasmid with the gene for the protein, or cDNA 2)
you know the base sequence around the site of
substitution An oligonucleotide primer that is
complementary to this region of the gene is
prepared with the substituted base(s) in place
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Site-Specific Mutagenesis
The two strands of the plasmid are separated and
the primer is hybridized The base pair
mismatches are tolerable at certain temperatures
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Site-Specific Mutagenesis
The primer is then elongated using DNA polymerase
and the double-stranded circle closed using DNA
ligase Subsequent replication creates two kinds
of progeny - one with the mutation and one
without Expression of the mutated plasmids
results in production of mutant protein
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Site-Specific Mutagenesis
Cassette mutagenesis is a variation on this where
a short sequence of DNA is substituted into a
plasmid - this results in a mutant sequence being
inserted into the protein product Designer genes
(novel proteins) can be created by splicing
together gene segments that encode protein domains