Analysis of DNA **Practice
-- It will be on the Quiz**
Southern, and Western blots
is gene technology? All gene
is based on microbial genetics--ways of doing in the test tube what
and viruses do naturally. Several of this week's gene manipulations are
exemplified by the article on overexpressed
angiopoietin in a transgenic mouse.
Electrophoresis Images and text based on MIT
molecules on the basis of their size.
The gel is set
electrophoresis in a tank holding pH buffer. Electrodes
an electric field:
of gel material,
usually agarose or polyacrylamide. The gel is a matrix of
forming sub-microscopic pores.
of the pores
can be controlled by varying the chemical composition of the gel.
molecules to separate
(DNA RNA) carry a net negative charge (why?)
they move along the electric field toward the positive
cathode. (To separate proteins, a detergent would be
included which coats the protein with negative charge.)
are held up as they try to pass through the pores of the gel, while the
smaller molecules are impeded less and move faster. This results
in separation by size, with the larger
nearer the well and the smaller molecules farther away.
on the basis of size (volume in solution),
which is not necessarily molecular weight.
Aside from the
the distance migrated is roughly proportional to
the log of the inverse of the molecular weight (the log of
Gels are normally depicted as running vertically, with the wells at the
top and the direction of migration downwards. This leaves the large
at the top and the smaller molecules at the bottom. Molecular weights
measured with different units for DNA, RNA, and protein:
molecules of the
same molecular weight will run differently if one is supercoiled,
the supercoils constrain the shape to be smaller.
molecules of the
same molecular weight will run differently if one has much
base pairing, making it "smaller."
run in one well of the gel are used to calibrate the molecular weights
of sample molecules. Below is a gel stained with a dye: a colored
molecule which binds to a specific class of macromolecules in a
manner (probes bind in a sequence-dependent manner).
Molecular weight is measured in base-pairs, or bp, and commonly in
(1000bp), or kbp.
Molecular weight is measured in nucleotides, or nt, and commonly in
(1000nt), or knt. [Sometimes, bases, or b and kb are used.]
Molecular weight is measured in Daltons (grams per mole), or Da, and
in kiloDaltons (1000Da), or kDa.
1 contains only one size class of macromolecule - it could be a
plasmid, a pure mRNA transcript, or a purified protein. In this case,
would not have to use a probe to detect the molecule of interest since
there is only one type of molecule present. Blotting is usually
for samples that are not complex mixtures. By interpolation, its
weight is roughly 3.
2 is what a sample of total DNA cut with a restriction enzyme,
cellular RNA, or total cellular protein would look like in a gel
with a sequence-independent stain. There are so many bands that it is
to find the one we are interested in. Without a probe (which acts like
a sequence-dependent stain) we cannot get very much information from a
sample like this.
are used for different classes of macromolecules. DNA and RNA are
generally stained with ethidium bromide (EtBr),
intercalating agent. The DNA-EtBr complex fluoresces under
UV light. Protein is stained with Coomassie Blue or Silver
In nature, DNA molecules
for various functions -- even DNA between different species. But
twenty years ago, despite the work of Barbara McClintock and others,
extent of this recombination was not appreciated. DNA was still
to be the "master molecule," not to be violated by "unnatural"
When scientists began to manipulate DNA in the test tube, many
feared that disastrous monsters would result, with unspecified dangers
to people. In 1977 scientists at the Asilomar Conference proposed
sweeping regulation on so-called "recombinant DNA," technologies which
recombine DNA from different species in the test tube.
Since then, the dangers
to be little more than those of "natural" genetic mixing.
we remain concerned about issues such as:
Engineering food crops
pesticides. The pesticide resistance genes can escape into
populations of weeds.
Engineering a human
such as E. coli, to produce a deadly toxin such as
In theory this could be done, although it's not clear where such an
would live, or how well it could "compete" with natural flora.
Societal dilemmas of
How far shall we use reproductive technology to shape future humans?
How do we manipulate these
processes for biotechnology; for instance, to make a bacterium that
large quantities of insulin?
One approach would be to
appropriate gene from human DNA and paste, or splice, it into a vector
such as a plasmid or phage DNA. Our "scissors" are the class of
called restriction endonucleases
An "endonuclease" is an
that cuts duplex DNA in the middle, not at an end (for
Different species of bacteria have evolved different restriction
each to cut foreign DNA that gets into their cells by mistake. To
be cut, the DNA has to lack their own pattern of protective
There are well over a hundred restriction enzymes, each cutting in a
precise way a specific base sequence of the DNA molecule.
DNA only at a specific site, usually containing 4-6 base pairs.
enzyme has to cut the DNA backbone twice, recognizing the same type of
site; therefore, the site "reads" the same way backwards as forwards--a
from two different DNA molecules can hybridize together; then the nicks
are sealed using ligase.
(Where does ligase come from? What is its natural function?) The
result is recombinant DNA.
this recombinant vector is inserted into E. coli, the cell will be able
to process the instructions to assemble the amino acids for insulin
More importantly, the new instructions are passed along to the next
of E. coli cells in the process known as gene
How can we use restriction
to analyze the plasmid products of ligation, and tell whether we in
have ligated the correct molecule:
Suggest several "incorrect" ways the plasmid could recombine.
Program. You MUST practice restriction analysis with this
program; it will be on the quiz and/or the test.
How do we get the
molecule into a bacterial cell? Usually by transformation
(for a plasmid) or by in vitro packaging
into a phage head coat (for a phage vector such as lambda phage).
The above is a highly
description of recombinant DNA technology.
How would we actually
the appropriately cloned gene? There are many different ways,
on the specific case. Here is one example, in which a partial
of the protein enables us to reverse the code and
determine an approximate DNA sequence to use for a radiolabeled
probe. The DNA probe will hybridize
to clones containing the correct DNA, even if it is just one piece cut
out of an entire genome.
et al, W. H. Freeman & Co., current edition
probe is made by determining a short
of the protein sequence, then "back translating" to the possible DNA
Short DNA sequences are synthesized to match the protein
Then these DNA oligomers (known as "oligos") are radiolabeled, and
to the blotted clones. They should hybridize only to clones
sequence encoding the desired protein.
Suppose we need to clone a
containing lots of introns. What will
when the bacterium tries to express it?
this problem, we can start with mRNA
isolated from tissues that produce the desired protein. We
then use reverse transcriptase enzyme
(produced by a retrovirus related to HIV) to reverse transcribe the
into a DNA molecule that now is free of introns. Now we can
"sticky ends" onto the cDNA
and recombine it into a phage or plasmid vector.
Know the differences between genomic cloning and cDNA cloning.
the relative ADVANTAGES and DISADVANTAGES of each technique--depending
on the aim of your research.
Summary of technique
a heat-stable DNA polymerase is used, most commonly Taq
Polymerase from the thermophilic microbe Thermus
aquaticus. Thomas Brock discovered T. aquaticus
from a hot spring at Yellowstone National Park.
an even more heat-resistant polymerase has been developed from a
microbe growing at 110 degrees C in hydrothermal vent ecosystems in the
deep ocean; it's called "Vent Polymerase."
Polymerase is put with the DNA to be amplified, plus all four NTPs,
two primers facing each other, about 200 - 6000 kb apart. (Why
do we need primers?) The primers are
selected based on the DNA region you want to amplify. The tube is
placed in a thermal cycler.
synthesized from each primer, for about 2 minutes. Then the
is raised to 95 degrees C -- enough to denature (split apart) the DNA
pairs. But the Taq Polymerase remains intact, because it comes
an organism that evolved to grow at this temperature.
temperature is decreased again, and primers again can hybridize to the
DNA--both the old AND the newly synthesized strands. Again, Taq
extends new DNA strands. Again, the temperature is raised.
cycles, the amount of DNA sequence between
the two primers increases exponentially.
First 2 strands, then 4, 8, 16, up to about a million. Thus, in a
couple of hours, you can get million-fold amplification of a DNA
et al, W. H. Freeman & Co., current edition
PCR has replaced cloning
many purposes, particularly the sequencing of DNA. It is faster
requires no vectors, which can mutate as they reproduce. It
can be used forensically, to amplify tiny amounts of DNA from criminal
evidence; or clinically, to detect DNA sequences linked to inherited
the microbial species of various environments in Knox County.
were placed directly into PCR reactions containing primers for
of ribosomal RNA genes (rDNA).
4 contains an
amplified band of the predicted size, 1000 bp. The top of each
contains genomic DNA. The smears at the bottom contain the PCR
DNA will be sequenced
and matched through GenBank to determine the microbial genus.
to Dan Nickerson
'00 and Adam Marks '01.
The main limitations of
For more information on PCR
Only relatively short
be amplified reliably. Anything more than 10,000 base pairs is
to be amplified.
You need to know the
sequences to use, at both ends of the sequence you want to
If two related genes have the same end sequences, you might amplify the
You only obtain a DNA
To see this DNA at work inside a living organism, some type of cloning
has to be done.
Technology - discussion of preparation of the sample, the master
and primers, and detection and analysis of the reaction products.
sequence analysis Modified from MIT
The sequence of DNA base
can be analyzed by
mapping. Construct a "road map" of
sites. A program to do this is WebCutter.
Analysis. Cut and clone various restriction
fragments, and determine the exact
of base pairs. All sequence information is deposited in GenBank.
If you just want the sequence of the peptide translated
from the RNA, you have to look for insulin mRNA
Once we have a piece of DNA
cloned, it is amplified (available in
copies) and we now have a living clone
provides, in theory, an indefinite source of the DNA sequence.
do we analyze the sequence?
DNA Sequence Determination
The fragments are
or enzymatically labeled. They can be separated on a gel, or on a
fluorescence analyzer. All published DNA sequences in the world
deposited in GenBank.
Once you have identified a
region of DNA of interest, you need to find out the precise sequence of
DNA nucleotides. This is done by di-deoxy
in which a DNA polymerase is put together with dNTPs in four different
reactions, each containing a small amount of one di-deoxy NTP (ATP,
CTP, or GTP). The di-deoxy nucleotide lacks a 3'OH to continue
extension, so the chain terminates. Each reaction produces a
of fragments terminated at A, T, C, or G.
images from Davidson
of dideoxy sequencing reactions were subjected to electrophoresis in
different lanes of a gel. Each lane contained a reaction using a
different dideoxy terminator nucleotide. The data looked
the image at right.
Image from your textbook, Freeman,
S. (2002) Biological Science.
products of all four
sequencing reactions are loaded in a single gel lane or capillary tube
and subjected to electrophoresis. Molecular labels consist of
dyes instead of radioactive nucleotides. The gel looks something
Image from the University
of Maine DNA Sequencing Facility.
DNA in the gel
lanes are read by a computerized fluorescence detection system that
the intensity of light emission from each "band." The final
output is a "trace" or "electrophoretogram" that plots the intensity of
different color emissions vs. the length of the DNA being
By observing the progression of peaks of different colors, the DNA
is derived (A C
G T). The processed data look
Image from http://www.qiagen.com/
How do we find genes of
in a genome? Or a particular mRNA in the total cellular
Or a particular protein out of all cell protein?
In solution, hybrid
complexes (usually called hybrids) of the following types can exist
combinations are possible):
There are two important
A single-stranded DNA molecule (ssDNA probe)
can form a double-stranded, base-paired hybrid with a ssDNA target if
probe sequence is the reverse complement of the target sequence.
A radiolabeled DNA probe can be applied to DNA from a gel transferred
a membrane, called a Southern Blot
(named for its inventor).
A single-stranded DNA (ssDNA) probe molecule can form a
base-paired hybrid with an RNA (RNA is usually a single-strand) target
if the probe sequence is the reverse complement of the target
An RNA can be radiolabeled to probe a Southern Blot; or, a ssDNA probe
can be applied to membrane-bound RNA, called a Northern
Blot (name is a pun on Southern.)
An antibody probe molecule (antibodies are proteins) can form a complex
with a target protein molecule if the antibody's antigen-binding site
bind to an epitope (small antigenic region) on the target protein. In
case, the hybrid is called an 'antigen-antibody complex' or 'complex'
short. A radiolabeled antibody can probe membrane-bound proteins,
called a Western Blot
(an even worse pun.)
These properties allow you
hybridization to perform a molecular search for one DNA molecule, or
RNA molecule, or one protein molecule in a complex mixture containing
reactions are specific - the probes will only bind to targets
complimentary sequence (or, in the case of antibodies, sites with the
reactions will occur in the presence of large quantities of molecules
not identical to the target. That is, a probe can find one molecule of
target in a mixture of zillions of related but non-complementary
Northern, and Western Blots. Link to summary.
are named for
the target molecule.
Southern Blot--DNA cut with
enzymes - probed with radioactive DNA.
Northern Blot--RNA - probed
radioactive DNA or RNA.
Example--used to measure angiopoietin angiopoietin
expression from cDNA in transgenic mouse.
- probed with radioactive or enzymatically-tagged antibodies.
These molecules must
then be immobilized
on a solid support, so that they will remain in position during probing
and washing. The probe is then added, the non-specifically bound probe
is removed, and the probe is detected. The place where the probe is
corresponds to the location of the immobilized target molecule. This
is diagrammed below:
In the case of Southern,
and Western blots, the initial separation of molecules is done on the
of molecular weight, by gel electrophoresis.
DNA is first
cut with restriction enzymes and the resulting double-stranded DNA
have an extended rod conformation without pre-treatment.
is single-stranded, RNA molecules often have small regions that can
base-paired secondary structures. To prevent this, the RNA is
extensive 2' and 3' structures and are not always negatively charged.
are treated with the detergent SDS (sodium dodecyl sulfate) which
2' and 3' structure and coats the protein with negative charges.
to Solid Support. After the DNA, RNA, or
has been separated by molecular weight, it must be transferred to a
support before hybridization. (Hybridization does not work well in a
This transfer process is called blotting and is why these hybridization
techniques are called blots. Usually, the solid support is a sheet of
paper (sometimes called a filter because the sheets of nitrocellulose
originally used as filter paper), although other materials are
used. DNA, RNA, and protein stick well to nitrocellulose in a
After a series of treatment
the probe is added. The probe hybridized to the target
is visualized either by autoradiography or by enzyme reaction.
The important properties of the three blotting procedures of DNA
put most of the protein-encoding genes onto a microarray chip, using
based on the DNA silicon chip industry. The chip can be used to
to cellular RNA, and measure the expression rates of a large number of
genes in a cell.
From "Everything's Great When
Sits on a Chip," The Scientist, Volume 13, #11, May 24, 1999
Animals have two different
of cells: germ line and somatic.
Only alterations in the
cells can be transmitted to future generations. However, some
of somatic cell gene therapy
can be useful in treating patients. For example, people with
fibrosis can receive the cloned CFTR gene in a nasal spray, which then
infects the lining of their lungs and improves lung function. The
infected (transformed) epithelial cells eventually are lost, however,
normal processes of tissue growth, so the treatment needs to be
There are two basic ways to clone a gene in the mammalian germ line.
cloned a valuable gene into an animal, say a sheep that makes insulin
its milk, how can we produce lots of identical progeny quickly without
sexual reproduction? This is what the popular press calls
(More after spring break.)
Inject a DNA fragment
your gene into the nucleus of a fertilized egg (germ-line
gene cloning) or of body tissues in a mature host (somatic
gene cloning). The DNA gets taken up at random somewhere
one of the chromosomes.
An example of germ-line
cloning is the injection of angiopoietin
cDNA into the fertilized mouse egg.
(An example of somatic
cloning is gene therapy for cystic fibrosis: inhale a vector containing
the CF gene, which gets incorporated into the DNA of cells lining the
Somatic cloned genes are not inherited by offspring.)
- Put a transgene
and negative selective markers into an embryonic
stem cell tissue culture. The ES
take up the gene by homologous recombination, replacing the host
The ES cells now are injected into a blastula of an unrelated host, and
some of the next generation progeny arise from the ES cells. To
more about this, read Capecchi's article
Targeted Transgenes, on reserve.
But when you create a
do you know it worked? You need to
transgene really incorporate into the genome? Where?
RNA expressed? (Or prevented from expression, by a null allele?)
a desired protein expressed?
Plants don't have separate
and germ cells, so creating transgenic plants is easier than creating
animals. Techniques include the use of microprojectile bombardment (the
"gene gun") and the use of Agrobacterium tumefaciens.
practice problems: Try problems 1-9 of the Recombinant
DNA Technology Problem Set from
the Biology Project at
Univerisity of Arizona.