Laboratory #5: Cheek Cell DNA extraction
With the work of scientists such as Hershey and Chase or Avery, McCleod and
McCarty, DNA became known as the genetic material. DNA is one type of nucleic
acid, and is a polymer made of repeating units called nucleotides. Each nucleotide
consists of three basic components, a phosphate group, a 5 carbon sugar known as
deoxyribose and a nitrogenous base. The backbone of the DNA molecule consists of
the phosphate group and the deoxyribose sugar. Located in the interior of the DNA
molecule are the nitrogenous bases, of which there are four Adenine (A), Thymine
(T), Cytosine (C), and Guanine (G).
To make a nucleotide, at its core is the deoxyribose sugar. Each carbon of the
deoxyribose sugar is given a specific designation, 1’, 2’, 3’, 4’ and 5’. The nitrogenous
base is bound to the 1’ carbon, the phosphate group is bound to the 5’ carbon, and
thus is called the 5’ phosphate. An OH group is bound to the 3’ carbon, and is thus
called the 3’ OH group.
A bond can form between the 5’ Phosphate of one nucleotide and the 3’ OH of
another nucleotide. This is known as a phosphodiester bond. When many
nucleotides are linked together in this manner, one can make a single strand of DNA.
When we look at the single strand of DNA, the end of the molecule with a free
phosphate group is the 5’ end (unbound), and the end with the free 3’ OH group
(unbound) is the 3’ end.
Each DNA molecule has a specific composition of nitrogenous bases with
respect to one another. The rules behind the composition of nitrogenous bases in a
DNA molecule were derived by an Austrian Biochemist Erwin Chargaff, in the early
1930’s, and are considered Chargaff’s rules. Chargaff proposed two rules; the first
rule is that the number of adenines is equal to thymines in a DNA molecule. The
second rule is that the number of cytosines is equal to the number of guanines in a
DNA molecule. Although these rules were absolutely correct, surprisingly, Chargaff
was never able to prove them experimentally.
Eventually, James Watson and Francis Crick were able to prove these rules by
determining the secondary structure of DNA was a double stranded double helix.
Therefore, to make a full DNA molecule, you must have two strands that are
somehow bound together. The two strands in the DNA molecule are anti-parallel.
Therefore, the 5’ ends of each strand are on opposite ends of the double stranded
molecule and the 3’ ends are on opposite ends of each molecule. Between the two
strands are located the nitrogenous bases. It is bonding between the nitrogenous
bases that allow for the two strands of the DNA molecule to be bound together.
Specifically, Watson and Crick found that adenine base pairs with thymine by two
hydrogen bonds, and guanine base pairs with thymine by three hydrogen bonds.
It is the sequence of nitrogenous bases within DNA that allows it to hold the
genetic information, and the sequence of each DNA molecule is depend on the
nitrogenous bases used for each nucleotide added to the chain. Since all of us are
humans, but appear slightly different, if you look at the sequence of our DNA, the
sequence should be the same except for a few slight differences.
In this laboratory, we will isolate DNA from human cheek cells, and then
visualize it. As a matter of fact, DNA can be found in the nuclei of almost all of the
cells present within the human body, with one exception being the erythrocytes (red
blood cells). The DNA housed within the nuclei is not just placed there in a
disorganized fashion. If it were, there would not be able to place all of the our DNA,
which consists of almost (300 billion base pairs-18,000 genes). Instead the DNA
must be packaged in an efficient manner within the nucleus. For humans, the DNA
is packaged into linear chromosomes. Specifically, we have 46 chromosomes, or 23
pairs of chromosomes. On these chromosomes consists of all the genes necessary to
produce our outward traits, and all the genes necessary for cells to fulfill all the
necessary functions to allow for life (for example-genes that encode enzymes
involved in glucose breakdown etc.)
To isolate the cheek cell DNA, we will collect our own cheek cells by gently
chewing our cheeks, and then removing our cheek cells by using a mouth-wash. The
cheek cells that you remove from your cheeks are still intact. Therefore, in order to
isolate the DNA from the cheek cells we will next lyse the cells by adding a lysis
buffer, which usually has some sort of detergent that breaks membranes. We will
also remove any proteins that are expelled from the cells by lysis using a protease
(an enzyme that breaks down proteins). Once the DNA has been removed the cells,
it will be in solution, and still cannot be visualized. In addition, the DNA will be in
solution with the rest of the materials. To visualize the DNA must be brought out of
solution by precipitation. We will precipitate the DNA using cold isopropanol. Once
the cold 95% ethanol is added, you will a stringy white material appear. This is your
DNA. In a real laboratory setting, the once the DNA is isolated and precipitated, it
can be used for such activities as forensic testing, genetic testing, it can be
sequenced, or used in other research pertaining to DNA. Some of these techniques
will be introduced at a later time in this laboratory.
Procedures for Isolation and Precipitation of Human Genomic DNA From
Cheek Cells
1. Obtain a 15 mL tube containing 3 mL of sports drink
2. Label the tube with your initials
3. GENTLY chew the inside of your cheek for about 30 seconds. Do not chew hard
enough such that you either hurt yourself or bleed. This dislodges cells from your
cheek making them free.
4. Take the 3 mL of water from your tube and rinse your mouth vigorously for 30
seconds (Do not swallow the water). This will allow you to collect all the cells you
dislodge from step 3.
5. Carefully expel all the water from your mouth back into the 15 mL tube.
6. Obtain the 2 mL tube labeled lysis. This contains the lysis buffer. This will act to
break open your cells and and liberate the DNA from the cells such that it ends up in
solution.
7. Pipet all 2 mL of lysis buffer to your tube containing your cells. Gently invert the
tube 5 times (with the cap on). At this time you are lysing your cells. Place any
observations you may have in your notebook.
8. Obtain the tube labeled Proteinase K. Proteinase K is an enzyme that acts to
break down proteins. The protease enzyme will break down all of the proteins in
your sample.
9. Add 5 ul of proteinase K solution to your sample and gently invert it 5 times to
mix.
10. Place your sample in the 50 C water bath for 10 minutes. Your protease enzyme
works best at 50 C.
11. Obtain a tube of cold alcohol. Holding your tube, add 10 mL of isopropanol into
your tube containing the DNA. This should precipitate your DNA.
12. Invert your tube 5 times to mike in the isopropanol
13. Add another 10 mL of cold isopropanol and mix by inversion again.
14. Add another 10 mL of cold isopropanol and mix by inversion again.
15. Let your tube sit undisturbed for 5 minutes, and observe what happens
16. After 5 minutes, slowly invert the tube again 5 times, and visualize the DNA
precipitating.
Procedures For Plasmid Preparation
1. Obtain 5 mL cultures for E. coli containing either pBR322 DNA
2. Pipette 1.5 mL of each culture into separate 1.5 mL microfuge tubes
3. Spin your microfuge tubes for 15 seconds to pellet the cells
4. Pipette off the supernatant and to your tubes containing the pellets, add 1.5 mL of
the appropriate culture to each
5. Spin your microfuge tubes again for 15 seconds to pellet the cells
6. Pipette off the supernatant
7. Place your tubes on ice
8. To each of your tubes add 100 ul of solution 1 and resuspend the pellets
9. Add 20 ul of 10 mg/mL lysozyme. Mix your tube and incubate at room
temperature for 2 mins.
10. Add 200 ul solution II (lysis buffer). Invert your tube at least 5 times to mix and
incubate on ice for 5 mins.
11. Add 150 ul solution III (neutralization buffer). Invert at least 10 times to form
white clumps. Incubate on ice for 5 minutes
12. Microfuge for 5 minutes
13. Add 400 ul phenol:chloroform (be careful not to splash this on you). Vortex 30s
and then microfuge for 2 minutes
14. Transfer aqueous (upper) phase to a new tube. Add 1 ml room temp. EtOH. Mix
well and stand at room temp for 2 minutes.
15. Microfuge 5 minutes in cold microfuge. Pour off EtOH and let pellet dry completely.
16. Resuspend pellet in 50 ul of sterile water and add 5 ul of RNase (this will degrade all
RNA in your solution)
17. Place tubes at 37 C for 30 mins.
18. Store in freezer for further use.