Applications of Biotechnology II: Study
and Characterization of DNA
DNA Fingerprinting and Genetic Screening
Separating Restriction Fragments - Gel
Electrophoresis
DNA fragments can be separated by size
on agarose or polyacrylamide gels. Agarose is a seaweed extract available
in powdered form. The powder is mixed with buffer, heated to dissolve
the agar, poured in a mold, and allowed to cool. As the agarose cools
a gel will form due to cross-linking of sugar polymers. The interior space,
at the molecular level, resembles a meshwork or spider web.
DNA molecules move through the network of fibers that
make up the gel more or less quickly based on size. Small DNA fragments
can move more quickly through the meshwork than larger fragments.
The gel is poured with a well-forming
comb in place. When the gel has solidified the comb is removed and the
DNA fragments are loaded in the wells. Before loading the DNA fragments
are mixed with a loading buffer that contains a mixture of dyes, glycerol,
and typically EDTA, a chemical that binds ions and inhibits the activity
of enzymes (nucleases in particular, enzymes which would degrade our DNA
fragments).
The gel is placed in a box which has electrodes at
either end. Buffer is added to cover the gel. When power is supplied
to the gel box current flows through the buffer. DNA, being negatively
charged, will migrate through the electrical field toward the anode.
In order to visualize the movement
of the DNA fragments through the gel a couple of tracking dyes are typically
included in the loading buffer; a low molecular weight dye that migrates
faster than the smallest DNA fragments, and a high molecular weight dye
that migrates a bit slower than the largest DNA fragments. When the smaller
dye reaches the end of the gel the power is turned off.
This separation allows DNA fragments containing the
area of interest (the gene to be cloned or area to be sequenced) to
be isolated. It also forms the basis of DNA fingerprinting techniques
(we'll talk about this later).
Since the DNA isn't visible
as it runs on the gel we need some way of visualizing the DNA after
we've separated the fragments.
One way is to stain the gel with ethidium
bromide, which will combine with the DNA strands and fluoresce orange
when excited with ultraviolet light.
If you just want to identify the band that has a
specific DNA sequence you would use a probe - just like we did with
colony hybridization.
This process is called Southern blotting.
RFLP
After scientists started performing electrophoresis on restriction
digests they noticed that the size and number of restriction fragments could
vary from person to person (or from organism to organism or between strains
of the same species).
Due to mutations, DNA molecules from different people may
lack some restriction sites, may have additional restriction sites, or may have
additions or deletions between restriction sites. Any of these things would
change the size and number of restriction fragments resulting from a restriction
digest. When the restriction digests are electrophoresed the distribution
of bands, or the banding pattern, will show the differences.
Strains of organisms that have different antigens and different
virulence factors may also show different banding patterns. Below is an
example of a DNA fingerprint that suggests the E. coli present in people who
drank contaminated apple juice was also present in the apple juice.
Southern Blotting
After running electrophoresis of DNA fragments you can stain
the gel and see all the bands present, but you can't tell if a particular band
contains a specific nucleotide sequence. Specific nucleotide sequences
you might be looking for include genes involved in diseases or sequences that
are used as markers - say repeating sequences that tend to vary in the number
of repeats present between people and could be used as identifiers.
There are other times when you might actually want to cut
the band that has the restriction fragment containing the labeled DNA out of
the gel.
In order to identify these sequences you just need to probe
the gel for the sequence of interest. It's a lot like colony hybridization
but is done with a restriction digest.
PCR
The polymerase chain reaction (PCR) is used to make
multiple copies of a desired piece of DNA enzymatically (in a tube without
a living organisms doing it for you).
All this requires is a template molecule ( a piece
of DNA that contains the area you want to make copies of, typically a
gene but often referred to as "the area of interest"), primers
that are complementary to sequences on either side of the area of interest,
deoxynucleotide triphosphates, and DNA polymerase. You know where
to get the DNA you want to amplify (your sample DNA), and you get your
deoxynucleotide triphosphates and DNA polymerase from a molecular biology
supply company. The question is where to get the primers.
You get your primers from the same place you got your
probes. You use your DNA synthesizer, or borrow someone else's,
or order the primers from someone who has a DNA synthesizer. Yes,
you have to know the sequence of areas on either side of the area interest
(a lot of DNA sequence data has been generated using random
primers, however).
Steps:
Denature target DNA with heat (94 - 95O
C for 1 minute) to separate the double stranded DNA template
Cool to a temperature that will allow the short
primers to anneal to the target DNA but isn't cool enough to allow the
template strands to reanneal (55-65O C for 1 minute)
Increase the temperature to an optimal temperature
for the polymerase to synthesize (72O C for 1 minute)
Repeat - each cycle produces 2 new copies from each
template strand so the number of copies increases exponentially with
each cycle - the number of copies can be calculated, assuming faithful
replication and an abundance of dNTPs and primers, as 2n
copies where n = the number of cycles
PCR can be used to increase the amount of DNA in samples
to detectable levels. This procedure is used for DNA fingerprinting, DNA
sequencing, construction of DNA probes, the diagnosis of genetic abnormalities,
identification and classification of bacteria, and the detection of viruses.
DNA Sequencing
There were a couple of different methods of DNA sequencing
developed in 1977. Allan Maxam and Walter Gilbert developed a method
based on chemical degradation of the bases which worked pretty well,
but Frederick Sanger developed a method that is still in use today and
forms the basis of some major advances in DNA sequencing.
The Sangar method was based on a DNA synthesis reaction
that used a mixture of regular deoxynucleotides and dideoxynucleotides
to produce a series of daughter DNA strands that ended prematurely at
each base in the template strand. It's probably easier to show you
how it works than to describe it. The important part of the process
was the use of dideoxynucleotides (or "terminators").
The way to make use of the terminators is to set up
a DNA synthesis reaction in a test tube and supply a mixture of deoxynucleotides
and dideoxynucleotides. You've got to use four different tubes,
and in each tube you put the same template, DNA polymerase, buffer, primers,
and deoxynucleotides, but only one kind of dideoxynucleotide.
In the example below the terminators are all dideoxyadenosine.
When the DNA polymerase reads a T on the template strand it will insert
either a normal deoxyadenosine (dA) or a dideoxyadenosine (ddA).
If DNA polymerase inserts a dideoxyadenosine synthesis stops and you get
a short strand. If DNA polymerase inserts a normal deoxyadenosine
synthesis continues.
The insertion of the terminators occurs randomly, so
the strands aren't necessarily generated in order, from the smallest to
the largest, but the point is that when the synthesis reactions are completed
you should have a complete set of newly synthesized strands containing
strands that terminate at each position adenosine is inserted by DNA polymerase.
Of course you set up a tube for each dideoxynucleoside,
so you have a ddA tube, a ddT tube, a ddC tube, and a ddG tube to get
strands that terminate at each base in the template. To visualize
the bands after you seperate them by electrophoresis you can label the
terminators or the primers.
Now run the four different reactions out
on a gel...
With the development of fluorescent dyes,
scanning lasers, and automated electrophoresis it bacame possible to do
all of the reactions in a single tube and run the resulting fragments
out in the same lane on the gel. The laser reads each fragment as it runs
through the gel and identifies the nucleotide at the end by the color
of the dye attached.
So you do your sequencing reactions, load
the gel, turn on the power, and go home.
Then you come in the next day and print
out your results.
Shotgun Sequencing
This is all great for sequencing relatively small areas of
DNA that contain genes of interest. An approach to sequencing the entire
genome of an organism that was employed by Craig Venter of Celera Genetics in
his "race" with NIH to complete the Human Genome Project is shotgun
sequencing.
Shotgun sequencing relies on digesting multiple copies of
an organism's genome with different enzymes to produce many overlapping fragments
which can be sequenced individually.
The sequence data from these fragments is analyzed by computer
to find overlaps. The fragments are then placed in order and the complete
sequence deduced.
The 'whole genome shotgun' method is applied to the entire
genome all at once, while the 'hierarchical shotgun' method is applied to large,
overlapping DNA fragments of known location in the genome.
Microarrays
Microarrays are a really cool way to rapidly screen samples
for the presence of specific genes and to look at gene activity (gene expression).
For example, gene mutations present in cancer or gene mutations that predispose
individuals to other diseases can be rapidly identified using microarrays.
Not only can microarrays detect the presence of a specific gene, they can also
offer quantitative data to determine whether there are multiple copies of a
gene present or to determine the level of gene expression (which genes in a
sample are turned on and just how many copies of mRNA are being transcribed
from those genes).
Microarrays are a set of DNA sequences arranged in a grid
on a substrate, like a microscope slide or membrane filter. The sequences
are typically the expressed sequences (cDNA) of genes of interest - for example,
let's say you wanted to analyze gene expression in yeast. You could set
up one yeast microarray that would have 6200 locations (one for each gene
in the yeast genome). Each location would contain multiple copies of the
gene assigned to that location. One experiment with this microarray would
be equivalent to running 6200 Southern blots.
You then hybridize the microarray with multiple DNA molecules
of unknown sequence isolated from the organism or tissue of interest.
The DNA molecules of unknown sequence are fluorescently labeled, so each location
on the microarray that hybridizes with a sample DNA molecule can be identified
by scanning with a laser and detecting the fluorescence emitted by the bound
sample DNA. As more sample DNA molecules bind to a single location the
fluorescence intensity increases.
