Techniques: DNA Extraction
Background
Deoxyribonucleic acid (DNA) is the hereditary molecule in all living cells. In
Prokaryotes, the DNA is present in a centralized area of the cell called the nucleoid. In
Eukaryotes, the DNA is present inside the nucleus. It is a double helix formed from two
antiparallel polynucleotide chains or strands made up of nucleotide subunits. A
nucleoide subunit consists of three parts: a phosphate group, a deoxyribose sugar and a
alternating nitrogenous base (Adenine, Cytosine, Guanine or Thymine). The phosphate
group of one nucleotide attaches to the sugar of the next nucleotide by a phosphodiester
linkage to make a polynucleotide chain. The bases obey the Watson and Crick rule of
base pairing: Adenine (A) pairs with Thymine (T), Guanine (G) pairs with Cytosine (C).
DNA resembles a ladder that is twisted so there is one turn for every ten rungs (or bases).
DNA forms very long molecules, much longer than the cell in which it is found. The
length of DNA can be calculated by multiplying the distance between bases, 3.4
Ångstroms (Å) (100,000,000 Å = 1 cm), by the number of bases in the chromosome,
which typically is 1 to 5 million for prokaryotes and about 3 billion for humans. For
prokaryotic microorganisms, which are only one or a few micrometers (μm) long, the
chromosomal DNA may be 1 millimeter (mm), or almost one thousand times longer than
the cell! In order to fit inside the cell, chromosomal DNA is packaged into coils and
supercoils.
While DNA can form very long molecules, the thickness of a DNA ladder is only 20 Å,
or about 1/50,000th the width of a human hair (50 to 150 μm). Because of its minute size,
individual DNA molecules must be visualized using an electron microscope, the DNA
must be coated with proteins and a heavy metal such as platinum. This coating thickens
the DNA strand and creates enough contrast between the DNA and the surrounding area
for it to be seen with the electron microscope (In the figure below, supercoiled and
relaxed DNA is shown employing electron microscopy; left to right, respectively).
DNA possesses the special property of replication, forming two identical progeny
molecules from a single parental molecule (this is the reason that it has evolved to serve
as the genetic material in cells). For this process, the two antiparallel DNA strands are
first partly denatured, or separated, by the action of proteins called DNA helicases. Then,
each DNA strand serves as the template for synthesis of the complementary strand using
Watson and Crick base pairing of nucleotide building blocks, utilizing an enzyme known
as DNA polymerase
A complication in this process is that DNA synthesis must have something to which it
attaches the first base pair in the DNA chain. So first, a short piece of RNA (11
nucleotides long) called a primer, is laid down by another enzyme, called primase. In
creating this primer, analogous Watson and Crick rules are followed, except that the
RNA base Uracil (U) is used in place of the DNA base T. The DNA polymerase extends
the chain of DNA from this primer. Later, the RNA primer is removed and replaced with
DNA by DNA polymerase.
Another twist in the DNA replication story is that the DNA chains are always synthesized
in one direction, called 5′ to 3′, and never in the opposite direction, 3′ to 5′ (Figure 2).
This leads to there being a leading strand in replication, which is formed using only one
RNA primer, and a lagging strand, whose synthesis requires many RNA primers (this
aspect of DNA replication is demonstrated in the figure below).
During replication, the accuracy of the DNA replication is checked by the editing activity
of DNA polymerase itself, as well as by a host of other repair proteins. In this way,
genetic information is faithfully transmitted from one generation to the next. (In the
figure below, when prokaryotic DNA is replicated the lagging strand is looped back on
itself in such a way that the leading and lagging strands are able to replicate in the same
physical direction and enzymatic directions).
Why would scientists want to extract DNA from cells? In research, DNA is used to study
fundamental aspects of organisms and their genes and to understand, diagnose and treat
genetic diseases. Isolated DNA may also be used to bioengineer cells for applications in
biotechnology. For example, bacteria can be engineered to produce proteins such as
insulin for diabetics. In addition, DNA is also used in forensic labs for genetic
fingerprinting, and is being used to exonerate innocent people and to convict criminals.
Extracting DNA from Wheat Germ
Wheat Germ cells (a plant cell) possess a tough cell wall to protect them from their
environment. DNA extraction requires breaking open the cell in order to extract the
DNA. Breaking down cell walls often requires harsh chemical or physical methods that
are technically challenging. The cell lysate that results from cell lysis contains a solution
of cell components including RNA, protein and most importantly for the purpose of this
lab, DNA.
As soon as the DNA is released, it becomes susceptible to breakage. Therefore, you
should handle the cell lysate gently. The more the solution is stirred and shaken, the
smaller are the resulting DNA fragments. If the fragments become too small, you will not
be able to spool them onto the stick. To precipitate the DNA so it can be wound onto a
spooling stick, you will add alcohol to the cell lysate. DNA is highly soluble in water, but
is insoluble in alcohol (such as ethanol and isopropanol) and in mixtures of alcohol and
water. Thus, as alcohol is added to the cell lysate, the insoluble components, including
the DNA, precipitate out of the solution. The DNA that precipitates is in the form of long,
fibrous molecules, which can be spooled by winding them around a stick. Remember, the
DNA you extract will be crude and will include a lot of proteins and RNA. If you wished
to analyze the DNA, it would have to be further purified (organic or chromatographic
extraction). Otherwise, the proteins and other impurities associated with the DNA would
inhibit the restriction enzyme digestion and or degrade the DNA.
Purpose
The purpose of this exercise is to isolate DNA from wheat germ cells and be able to see it
as a flexible, long thread. Isolated DNA can then be used for analysis through
conventional agarose gel electrophoresis or cloned for specific genes.
Materials per team
Water Bath (set to 60oC)
Large Test Tube & Rack
Weigh Paper
Eyedropper
1.5 mL Centrifuge Tube
P200 Pipetman
Extraction Solution (Detergent and salt in water)
Cleaning Brush
Cleaning Detergent
Electric Balance
Raw Wheat Germ
Ice Cold 95% Ethanol
Test Tube Rack
10 mL pipet
Stationary Centrifuge
Yellow Pipetman Tips
Procedures
(Adapted from DNA Extraction from Wheat Germ; Genetic Science Learning Center at The
University of Utah. http://gslc.genetics.utah.edu/units/activities/wheatgerm)
1. Pre-heat the prepared extraction solution to 60oC in the water bath.
2. Clean a large test tube with a test tube cleaning brush and liquid detergent. Rinse
thoroughly with tap water.
3. Place a diagonally folded weigh paper (or just a piece of paper) on the electric balance.
“Zero” the scale. Weigh out 1 gram of raw wheat germ.
4. Pour the wheat germ into the clean test tube.
5. Add 20 mL of the hot (60oC) extraction solution to the wheat germ and mix (by gentle
swirling) every 30 seconds over a 10 minute period. Try to avoid foaming.
6. Use an eyedropper or a paper towel to remove any foam that forms on top of the
solution.
7. Hold the test tube at eye level and tilt it at an angle. VERY SLOWLY pour 14 ml of 95%
ethanol down the side of the tube so that it forms a layer on top of the solution.
8. Gently place the test tube in a test tube rack and let it sit for 15 minutes. A white,
stringy, slimy DNA precipitate will form at the solution-ethanol interface; it may float
up to the top of the ethanol layer. DO NOT DISTURB THE LAYER!
9. Affix a yellow pipetman tip to a P200 pipetman and draw up a 200 L volume of
precipitated DNA within the ethanol layer (the instructor will show you how).
Dispense the volume into a 1.5 mL centrifuge tube.
10. Centrifuge the tube at high speed (remember to balance the centrifuge) for 2 minutes.
11. Look at the pellet at the bottom and the side of the tube – you have DNA (and
something else, remind the instructor and we will discuss it in class).
WORKSHEET
DNA Extraction
1. From where and why do scientists extract DNA? Think of some examples.
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2. Where in the eukaryotic cell would you find the most DNA? ____________________
What is the function of DNA? ____________________________________________
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3. The subunits (or building blocks) of DNA are called ____________________.
4. The components of the subunit are _____________________, __________________
and ___________________________.
5. The four types of nitrogenous bases present in a DNA molecule are:
______________________
______________________
______________________
______________________
6. What are the base pairing rules? ____________________, __________________.
7. Explain the effects of detergent/NaCl and blending to the cell.
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8. What is the purpose of adding cold ethanol?
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9. Two strands of DNA are linked together by _______________________ bonds.
10. DNA has approximately how many base pairs per turn?
11. If DNA has 3,400,000 base pairs how many turns does it have? _________________
12. If the base sequence of a strand of DNA is 3’AACGTTGCATCG 5’ then the
complementary strand should have a base sequence of ________________________.
13. DNA synthesis can proceed only from the ___ end to the ____ end.
14. ______ _________________ is the enzyme responsible for assembling the DNA
nucleotides complementary to the old DNA strand (new DNA synthesis).
15. Synthesis of which strand is continuous? _______________. Synthesis of which
strand is discontinuous? ________________.
16. An RNA primer contains about how many bases? ________. Which enzyme is
responsible for synthesizing RNA primer? ______________. RNA primer is
removed by which enzyme? _____________________.
17. Which enzyme is responsible for connecting the okazaki fragments by forming the
phosphodiester bonds? _______ _________________.
18. A. Give the nucleotide sequence of the newly synthesized DNA strands
B. Identify which is the leading and the lagging strand.
5’
T
A
A
G
T
C
A
T
C
G
G
C
T
T
T
A
A
A
A
T
A
T
T
A
3’
A
T