 |
|
|
Background:
DNA is isolated from the rest of
the cell contents by taking advantage of the different chemical
and physical properties of the molecules found within the cell.
Let's take a moment to review cellular structure. Where is DNA
located within the cell? What other chemical compounds are found
in a cell? How is the cell compartmentalized?
Each organism is different and the
cells of each kind of organism often contain special compounds,
which must be extracted away from the DNA. In the case of Echinocereus
cacti, the cells of the tissue contain MUCILAGE!!! Mucilage (often
used as glue!) is a very sticky, jelly-like substance whose function
in the plant cells is not known for certain, but it is thought to
act as an antifreeze. Chemically, mucilage is a highly complex
polysaccharide - very big, linear molecules, which have the ability
to form large complexes with other large molecules such as DNA.
The mucilage is contained within the cytoplasm of the cell, while
the DNA is contained within the nucleus (and within mitochondria
and chloroplasts). To isolate DNA from the tissue of Echinocereus,
the mucilage must be prevented from coming into contact with the
DNA and complexing with it. In order to accomplish this, the DNA
must remain within the nuclei while the mucilage is released from
the cell and then poured off. This procedure requires the use
of some dangerous and toxic chemicals, so we won't attempt to
do this in a high school classroom. However, we would like you
to have the experience of isolating DNA. Therefore, we will perform
a DNA isolation from the tissue of a plant that is not so difficult
to work with.
In addition to removing carbohydrate
compounds like mucilage, the DNA isolation procedure must also
separate the DNA from compounds like lipids (fats) found in cell
membranes and proteins (enzymes and structural components) which
are found throughout the cell. The DNA isolation procedure makes
use of the fact that lipids and proteins are soluble in chloroform
and that the DNA is soluble in water (aqueous) solutions.
DNA Isolation Protocol
1. Mechanically break open the cell
walls and membranes by grinding the tissue in a coffee grinder.
The plant tissue should be frozen before grinding and ground in
the coffee grinder with dry ice. Keeping the tissue frozen prevents
enzymes in the cells from breaking down the DNA. [CAUTION:
Do not touch the dry ice as it may cause "cold burns."]
2. Place the pulverized tissue into
a 50 ml centrifuge tube containing about 10 mL of CTAB buffer
solution. The buffer solution contains compounds which disable
the enzymes which will break down the DNA and also a compound
called CTAB which will combine with the polysaccharides and proteins
and chemically alter them. Mix the solution well to suspend the
cellular material. Incubate the solution at 60°C for at least
30 minutes.
3. Pour an equal volume (the same
amount as is already in your tube) of chloroform into the 50 mL
tube. "Extract" the solution by tilting or rocking the
tube gently back and forth (if you mix the solution vigorously
you may break your DNA molecules!). Chloroform is an organic compound
and all of the cellular compounds which are soluble in chloroform
(lipids, proteins) will be dissolved into the chloroform. DNA
is not soluble in chloroform and will remain dissolved
in the aqueous (water) layer. [CAUTION: Chloroform must
be used in a fume hood so that vapors will not be inhaled.]
4. Spin the solution in the centrifuge
for 5 minutes. The gravitational forces in the centrifuge will
completely separate the two phases according to density. Chloroform
is very dense and will be the bottom layer, while the water layer
is less dense and will be on the top.
5. Transfer the supernatant (the
aqueous layer containing the DNA) to a separate tube with a pipette.
6. Add 2/3 volume of ice-cold isopropanol
to the supernatant. The DNA dissolved in the supernatant will
not be soluble in the ice-cold alcohol and will start to precipitate
out of solution over a period of a few minutes. After a few minutes,
gently rock the tube back and forth (if you rock or shake
your tube hard you will break your DNA molecules). The
DNA should appear as whitish strands to clear jelly-like strands
which will float in the solution.
| Science Education Connection
Department of Biochemistry The University of Arizona May 1, 1997 warder@u.arizona.edu
http://biology.arizona.edu/sciconn/lessons/alongi/ |