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• Recombinant DNA technology
– DNA has now become the easiest of the cellular
macromolecules to be analyzed
– possible to cut out a region of DNA containing a
specific gene, to produce virtually unlimited number of
exact copies, and to determine its nucleotide sequence;
to alter or redesign the gene and transfer it back into
cells in culture or insert it into animals and plants.
– Provides a means to study the function of proteins and
their domains
– Used to detect mutations that are responsible for
genetic diseases, in forensic science to identify or
acquit suspects, to produce pharmaceuticals like insulin
for diabetics.
Restriction nucleases catalyze the
hydrolysis of a phosphodiester
bond in a nucleic acid at particular
site, determined by a short
sequence (often palindromic).
Most were isolated from bacteria
and are named for there source.
• A given restriction nuclease will always cut
a given DNA molecule at the same sites
resulting in the same set of DNA fragments.
• The sequence at which each restriction
nuclease will cut is short, usually 4-8 base
pairs.
– Hae III cuts at a sequence of 4 nucleotide pairs.
This would be expected to occur purely by
chance about 1 out of 256 nucleotide pairs ( 1
in 44) probability is 1 in 4n
Gel electrophoresis separates
DNA fragments of different
sizes.
Agarose or polyacrylamide gels
contain a microscopic network of
pores. Voltage is applied and
negative DNA travels toward the
positive electrode.
Visualized by soaking gel in a
dye that binds DNA and
fluoresces under ultraviolet
light.
Visualized by labeling DNA
with radioactive phosphorus
atoms prior to isolation, ex.
replicating virus in the
presence of 32P autoradiography
Larger fragments move more slowly,
held back by the gel’s matrix
Construction of physical maps of DNA includes charting the position of
various landmarks present along it, including known genes and
restriction sites. Physical maps created by treating DNA with different
combinations of restriction nucleases are called restriction maps.
This is a restriction map of the two alpha globin gene (red) cluster. Each
restriction enzyme used is designated by a letter.
Enzymatic method of sequencing DNA
excess
many reactions result in many
fragments ending in A, each a
different size depending on where
the ddATP happens to encorporate
Determining the
complete sequence of a
DNA fragment:
added
denatured to ssDNA
Complete sequences of several bacterial genomes, Drosophila (fruit fly),
and C elegans (worm) have been achieved in this way. Human genome
is being sequenced. This is a representation of the complete nucleotide
sequence of the genome of S. cerevisiae.
Denaturation and renaturation (hybridization) of DNA
Allows detection of specific sequences in DNA or RNA
DNA hybridiqation facilitates
diagnosis of genetic diseases
including prenatal diagnosis. This
requires a probe, a short singlestranded DNA (oligonucleotide,
10-1000 nucleotides long). These
(up to 120 nucleotides) can now be
synthesized using a machine.
If probes are short (20 nucleotides)
they can be hybridized at
temperatures at which only
perfectly matched DNA be stable.
southern blot
Who is a
carrier,
who is
effected?
Detection of specific DNA fragments by gel-transfer
hybridization (southern blotting). Alkali solution is sucked
up through the gel and nitrocellulose paper, into the paper
towels. It denatures the DNA and transfers the single
stranded fragments from the gel to the nitrocellulose sheet
where it firmly adheres.
In situ
hybridization to
locate genes on
chromosomes.
These are
metaphase
chromosomes.
Chromosomes are
briefly exposed to very
high pH to separate the
two strands.
Nucleotide probes
have been chemically
labeled and are
allowed to bind to the
chromosomes.
Fluorescent antibodies
that bind the chemical
on the probe are used
to visualize the
location of that
particular sequence.
In situ hybridization can also reveal the distribution of a particular
mRNA in cells in tissues. This is a group of cells in a growing
shoot tip of a snapdragon. Only a few cells express the mRNA for
cyclin, a protein that triggers cells to divide. The probe has been
linked to an enzyme that produces a dark blue reaction product
when a substrate is added.
formed by Eco RI
formed by Hae III and Alu I
Formation of recombinant
DNA molecules in vitro
Bacterial plasmids can be used to
clone DNA (make identical copies
of).
fragment to be cloned is cut by the
same restriction nuclease.
can this fragment go
in the plasmid
“forward” and
“backward”?
Cloning of a DNA fragment
How do we clone a
human gene (ex. – factor
VIII, mutated in
hemophilia A).?
1. break up the total
genomic DNA into
smaller more manageable
pieces – create a DNA
library. Since this is the
entire human genome is
is called a genomic
library. Many genomic
libraries for many
different organisms exist
and are shared between
researchers.
at a concentration
which leads to no
more than one
plasmid per
bacteria. Why is
this important?
How do we find the gene for Factor VIII? We don’t know
the sequence. One thing to do is purify some protein from
human blood donors. A small portion of this protein was
then sequenced. The genetic code was applied in reverse.
Probes are prepared by chemical
synthesis.
Go back to the living
colonies and pick up the
ones positive for the the
probe.
• This may lead to the cloning of only part of the
gene, as happened for Factor VIII.
• Often more work has to be done to piece together
the whole gene, introns and exons.
• Can you think of a way to take the DNA sequence
you have and “go fishing” for all the exons that
encode Factor VIII without the introns in the way?
• A gene can be large due to introns, it may be
advantagous to have the sequence of the exons
only. Where in the cell will you find this?
It is relatively easy to
create a cDNA
library.
Since Factor VIII is
made in the liver,
cDNA from liver
tissue was used to
isolate the mRNA
sequence by using
the DNA fragment
we cloned in the last
figure.
• Several important differences between a genomic
library and cDNA libraries.
– 1. genomic = same sequences regardless of cell type
cDNA = only sequences that are expressed
– 2. genomic = contains large amount of repetitive
DNA, introns, gene regulatory regions, spacer
DNA in addition to exons.
cDNA = contain only coding sequences and only those for
genes that are transcribed in this particular tissue or cell type,
even giving an indication of the level of transcription
– 3. Only cDNA contains uninterrupted coding sequence
of the gene. Can be used directly for expression in
bacterial or yeast cells which can not remove
mammalian introns. Can be used directly to deduce the
amino acid sequence.
genomic
cDNA
Different hybridization conditions
allow less than perfect DNA
matching.
New genes may have arisen
during evolution by duplication
and divergence of existing genes
and by using gene segments
(domains) from one gene in new
combinations (as discussed in the
last chapter). Most genes have a
family of related members often
with related function. Finding
these genes, or a gene from one
species in another, can be done by
differential hybridization using
sequences from the known gene
as DNA hybridization probes
for the unknown genes.
Polymerase chain reaction (PCR) is now quicker and less expensive
thatn the cloning methods we have already discussed. However, you
must have the beginning and ending sequences in most cases.
cooling in
presence of
large excess of
two DNA
primers
DNA polymerase used is isolated from a thermophilic bacterium.
It is stable at higher temperatures than eucaryotic DNA
polymerases. It does not need to be added at each cycle, it
remains active through the heating cycles.
Extra
Each cycle doubles the amount of DNA synthesized. A single cycle
takes about 5 minutes and the whole process is now automated.
PCR is very sensitive, it can detect a single copy of a DNA sequence
in a sample.
PCR can be used to obtain a
genomic or cDNA clone.
Because of its sensitivity,
this can be used to
determine what mRNA a
cell is expressing,
sometimes an indication of
protein expression. This
technique is not
quantitative.
PCR can be used to detect the presence of a viral genome in a
sample of blood.
PCR can be used in forensic science. DNA from a very small
sample of blood, skin, saliva, hair, or semen. The DNA sequences that create
the variability used here contain
short, repeated sequences such as
GTGTGT..., which are found in
various positions in the human
genome (VNTR). The number of
repeats in each run is highly
variable in the population. The
length of each fragment depends
on the number of repeats in this
area.
variable number of tandem repeat
DNA bands are
obtained from a set
of different PCR
reactions, each that
amplifies DNA from
a different VNTR
locus.
As you do more
separate VNTRs on
each sample the odds of
coming up with a
match become virtually
impossible.
DNA engineering: New
DNA can be made from
the combination of
naturally occurring
DNA sequences or
chemically synthesized
DNA.
cloned to purify and
amplify the new
DNA
cloned to purify
and amplify the
new DNA
cloned to purify
and amplify the
new DNA
Rare cellular proteins can be made in large amounts
for study and for clinical.
An expression
vector is used. These cloning vectors include
appropriate gene regulatory and promoter DNA
sequences necessary to enable an adjacent proteincoding DNA insert to be efficiently transcribed in
cells. This can then be translated in the cell.
Different cells require different regulatory sequences.
Some proteins require modification, and therefore
must be expressed in eucaryotic cells. The vector can
be inserted into the cell (transient expression) or
actually incorporated into the genome. Bacterial,
yeast, insect, or mammalian cells are used. The
protein can be easily purified after lysis of the cells or
it can be secreted.
Often, small amounts of a protein is isolated. In order to
study it, a short partial amino acid sequence is obtained.
This is used as a probe to screen a genomic or cDNA
library.
Once the gene or cDNA is cloned, it can be inserted into
an expression vector and introduced into a cell line for
large scale production.
What does a protein do? One way to
look at this question is to mutate the
protein. Mutants can be made
temperature-sensitive, so that the
mutation only shows when the
temperature is increased (or decreased).
Now, instead of starting with a random
mutation, we can start with a cloned gene
and make mutations in vitro. We can
introduce precise mutations, change one
specific amino acid. This is sitedirected mutagenesis. This can
be used in single cells and by
creating organisms which express
this particular mutation.
The “ultimate test” of the function of a gene mutated is to insert it into
the genome of an organism (transgenic) and see what happens. NOT
ULTIMATE! What problems do you see?
Transgenic techniques make it possible to produce complex
organisms that are missing certain gene products entirely, express
the protein only in certain cells, or express a mutated form of the
protein.
embryonic
stem cells
homologous
recombination
Transgenic animals often are
not viable due to the
importance of the knocked
out protein for development.
Here, a gene knocked out of
both chromosomes leaves the
developing mouse with no
cerebellum.
This is a common problem
when you are looking for the
function of a protein in an
adult mouse – redundancy.
Assume that defects in a hypothetical gene, X, have been linked to antisocial behavior. Two
copies of a defective gene X predispose a child to bad behavior from childhood, while a single
Chapter 10: DNA Technology 133
ddA ddC ddG ddT copy
of the gene seems to produce no symptoms until adulthood. Since the effects of the
gene
can be counteracted if treatment is started early enough, a program of voluntary genetic testing
is being carried out with delinquent prospective parents. Charles S. and Caril Ann F. have
been arrested on charges of robbery and assault, and Caril Ann is pregnant with Charle s’s
child. You obtain DNA samples from Charles, Caril Ann, and the fetus, and on each you per-form
two Southern blots using Not I to cleave the DNA and two oligonucleotide probes, A and
B, that hybridize to different
parts of the normal gene X, as
shown in Figure Q10–9A. You get
the results shown in Figure
Q10–9B.
(A) Which of the three individuals
have defects in gene X?
(B) Which individuals have a sin-gle
defective gene and which
have two defective copies of the
gene?
(C) Indicate the nature (single
base-pair mutation or deletion)
and location of each individua l’s
defects on gene X.
Q10–9