Fescue Chloroplast Genome Project - Part I
Lab 2120 Chapter 12 addendum
Extraction of Plant Total DNA and PCR amplification of
Chloroplast DNA
DNA Extraction Demonstration
Goal – Extract and purify DNA from living plant tissue.
Materials
 Plant tissue
 Liquid Nitrogen
 Urea Extraction Buffer
 Phenol:Chloroform (Very Dangerous)
 Isopropanol
 Ethanol
 TE resuspension buffer
Procedure
1. Grind Tissue in liquid nitrogen
Plant cells are surrounded by a tough cellulose cell wall (How tough? Cellulose is
the primary component of wood!). This means extreme measures must be used to
break open the cells. Nitrogen gas liquefies at -160°C (-320°F). When plant cells
are cooled to this temperature they become very hard and brittle enabling us to
grind them into a powder with a mortar and pestle.
chloro
plast
nucleus
mitochondrion
mitochondrion
chloro
plast
Figure 1: Diagram of a plant cell showing organelles where DNA is housed.
2. Suspend the Ground Up Tissue in Urea Lysis Buffer
Lysis buffer = Urea – denatures protein
NaCl – salt increases solubility of nucleic acid
Tris, ph 8.0 – pH buffer
SDS – detergent to denature lipid membranes and protein
EDTA – magnesium chelator, removes free Mg+2 from the solution.
Mg+2 is required by nucleases which break down DNA.
Water – Used to bring the solution up to volume. DNA is water soluble.
The frozen powder is made of broken and crushed plant cells. Every molecule
and macromolecule that makes a plant is mixed up in the powder. The challenge
is to separate the DNA in the mixture from everything else (protein, cellulose,
lipid, sugars, other organics like alkaloids and flavonoids). When we add
compounds like urea and SDS to our mixture they react with proteins and lipids
rendering them insoluble in water (aka hydrophobic). Meanwhile the DNA is
unaffected.
3. Extract with Phenol and pellet insoluble material
We now need to remove all of the insoluble molecules we created in step 2.
Phenol is an extremely nasty organic solvent that is great at attracting and
dissolving hydrophobic compounds. When we add this to our water based lysis
buffer we get two distinct layers (upper aqueous layer and lower organic layer).
The hydrophobic lipids and proteins will be removed leaving soluble nucleic
acids (DNA and RNA), sugars, and some water soluble organic compounds in the
aqueous layer.
4. Precipitate with Isopropanol
The water soluble material in the upper layer is removed from the hydrophobic
organic bottom layer and put into a new tube. The hydrophobic layer is
discarded. We now extract the DNA from the other water soluble molecules by
adding alcohol. When the solutions are mixed the DNA can no longer remain in
solution and clumps together.
Tube with aqueous
extract and alcohol.
White/opaque
blob is nucleic
acid
5. Suspend the DNA in TE buffer
TE buffer = Tris, ph 8.0 – pH buffer
EDTA – magnesium chelator, removes free Mg+2 from the solution.
Mg+2 is required by nucleases which break down DNA.
Water
Your mostly pure DNA can now be resuspended in an aqueous buffer and used
for experiments.
Polymerase Chain Reaction (PCR)
Introduction
Polymerase Chain Reaction harnesses the natural process of DNA replication to replicate
a specific piece of DNA and simultaneously exponentially amplify it. We will be using
this technique to take a piece of DNA out of its genomic context and prepare it for
sequencing.
How DNA replication is mimicked in vitro (in a test tube) during PCR.
1. Denaturing Step - When DNA is replicated the double stranded helix must first be
denatured. In nature (in vivo) this is accomplished with enzymes. In PCR the DNA is
heated.
2. Annealing Step - DNA polymerase will only initiate new strand synthesis if there is a
primer attached to the template strand. In PCR, the annealing is facilitated by rapidly
cooling the reaction thus allowing matching strands of DNA to find and bind to one
another.
3. Extension step - Once a primer is in place DNA polymerase can begin synthesis of the
new strand. The next step is to heat the reaction to the optimal reaction temperature of
the enzyme. In PCR we use a DNA polymerase extracted from bacteria which live in hot
pools such as those in Yellowstone National Park. This enzyme is very tough and can
withstand the high temperatures needed to denature the DNA in step 1. By the same
token, the optimal working temperature of the enzyme is also very high.
When the enzymes have processed complete new strands we end up with two pieces of
DNA when we began with one. Every time we repeat the denaturing/annealing/extension
steps we theoretically double the amount of DNA in our reaction tube. Our reaction will
undergo 30 of these cycles.
Goal – Amplify a portion of a monocot chloroplast genome.
Materials
 Plant DNA
 Synthetic DNA primers
 Thermostable DNA Polymerase
 Deoxynucleotides (Adenine, Thymine, Cytosine, Guanine)
 Reaction Buffer
MgCl2 – necessary DNA polymerase enzyme cofactor
Tris - pH buffer
KCl - important for DNA solubility and primer annealing
Procedure
1. Pipette the reaction components into a 0.2ml thin walled PCR tube.
0.5 μl of template DNA
2.0 μl of primers
2.5 μl of dNTP’s (10X stock)
2.5 μl of Reaction Buffer (10X stock)
0.2 μl of thermostable DNA polymerase
17.3 μl water added to bring volume to 25ul
In our lab exercise all components with the exception of the primers have been
pre-mixed. Each lab group will pipette 23μl of reaction mix into a PCR tube and
then add 2μl of the assigned primer set. Write your primer set in the box below.
My Primer Set
=
The primers have been designed to recognize and amplify a small portion of the
maize chloroplast genome which was completed in 1998. Since the primers
recognize portions of the chloroplast genome that are putatively important for all
plants they will probably also amplify portions of unsequenced DNA from other
plants. Write the plant species you are amplifying DNA from below.
My Plant
=
2. thermal cycling
Once the reactants are mixed the tubes are capped and put into the thermal cycler.
The automated thermal cycler is programmed to heat and cool the samples to the
temperatures indicated below and hold them for the prescribed period of time.
95°C 30 secs
50°C 1 min
68°C 5min
repeat 30 times
(denatures DNA into single strands)
(allows primers to anneal to their target sequences)
(optimal temperature for the DNA polymerase)
Chapter 12 addendum Laboratory report
Why did your instructor grind the plant tissue in liquid nitrogen?
Where in a plant cell do you find DNA?
How was it possible to specifically isolate DNA from the complex mixture of
biomolecules found in the powdered plant tissue?
The special DNA polymerase is isolated from bacteria that live in hot pools near the
boiling point of water. Why would you use this enzyme for PCR reactions?
If you start with a single double stranded molecule of DNA, how many copies would you
expect to have after two rounds of PCR?
Three rounds?
Four rounds?
Ten rounds?
Thirty rounds?