Download Note 8.2 - DNA Sequencing

UNIT 3: Molecular Genetics
Chapter 8: Genetic Technologies
pg. 364 - 421
8.2: DNA Sequencing
pg. 376 – 385
DNA sequence is amplified by PCR, it is then separated and
purified. Gel electrophoresis uses the physical and chemical
properties of the DNA sequence to separate it into fragments.
The gel electrophoresis uses an agarose gel to act like a sieve to
separate nucleic acids and proteins by the rate of their migration
through the gel. The gene fragments are usually negatively charged
and migrate towards the positive electrode.
The smaller fragments will migrate at a faster through the pours of
the gel, then their larger fragment counterparts. This causes the
separation of fragments.
Figure 3: The four steps in the separation of DNA fragments by agarose gel
electrophoresis.
There are 4 steps used to perform a gel electrophoresis;
Step 1: the agarose gel is prepared and placed in a gel box between
two electrodes. The gel is prepared with a series of wells, inset, at
one end of the gel. A buffer solution is poured over the gel to allow
the charge to pass from one electrode to another.
Step 2: Load the DNA sample solutions, PCR products, into the
wells using a micropipette. The first well is load with DNA marker
which acts as measuring scale to compare DNA migrations to. The
size of the DNA fragments migration distances are compared to the
markers to determine fragment sizes.
A loading dye is used to keep the DNA within well in the gel. Also
the dye moves a little faster then the DNA fragment. This allows
you to see the pace of the electrophoresis.
Step 3: The electric current is applied to the gel. The DNA
fragments that are negatively charged start to migrate towards the
positive electrode. Shorter DNA moves through the gel at a faster
rate then the larger fragments. Once the electric current is turned
off the fragment distance are compared to the markers. Distance
traveled will determine the size of the fragments.
Step 4: DNA fragments are hard to see therefore a stain
(Ethidium bromide) is applied to the gel staining the DNA
fragments, which are easily seen under an ultra violet light.
Gel electrophoresis: is a method for separating large molecules,
such as DNA, RNA, and proteins.
Molecule Marker: is a fragment of a known size that is run as a
comparison standard for gel electrophoresis.
Ethidium Bromide: is a large molecule that resembles a bas pair,
which allows it to insert itself into DNA; used for staining
electrophoresis gels.