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Ephraim Mansour
BCH 3033L U02
Yadira Reynaldo
31 March 2014
Cytochrome B DNA Isolation from Gallus gallus Liver
I ntroduction

Deoxyribonucleic Acid, DNA, encodes the genetic instructions used in the development
and functioning of all known living organisms and many viruses (Raven 2013). It is made up of
nucleic acids, proteins, and carbohydrates (Raven 2013). Nucleic acids are polar and as such are
soluble in polar solvents and are precipitated by non-polar solvents (Nelson et. al. 2008). In
eukaryotes DNA is located in the nucleus, mitochondria, and chloroplasts (Raven 2013). DNA in
the nucleus is double stranded linear whereas in the chloroplasts and mitochondria it is double
stranded and circular (Raven 2013). The purpose of this experiment is to isolate and characterize
DNA from a biological sample and amplify the cytochrome-b gene.
Classical isolation and purification of DNA consists of four steps. First, cell lysis is
necessary to release DNA (Nelson et. al. 2008). Next, enzymes that hydrolyze DNA are
deactivated (Nelson et. al. 2008). Then, proteins are dissociated and denatured (Nelson et. al.
2008). Lastly, the solvent is extracted and the DNA is precipitated out (Nelson et. al. 2008).
A pure preparation of DNA or RNA should have a ratio of Abs
260/280
of 1.8 to 2.0
(Esposti et. al.1993). Contamination with protein will lower this ratio. A kit is used to isolate
DNA to be examined under the UV spectra and a template is used to amplify a portion of the
cytochrome-b gene by polymerase-chain-reaction (PCR).
Polymerase Chain Reaction occurs via DNA polymerases and is used to amplify samples
of DNA (Raven et. al. 2013). This is especially useful in cases where there are small amounts of
sample. PCR reactions rely upon the heat stability of DNA polymerases from extreme
thermophilic bacteria. The classic DNA polymerase came from Thermus aquaticus (Taq) a
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bacterium isolated from a hot-spring (Raven 2013). Taq polymerase is relatively stable to boiling
allowing reaction mixtures to be heated to 95
o
C in order to convert ds-DNA to ss-DNA without
destroying Taq polymerase. The primers used are chemically synthesized single stranded
oligonucleotides that are designed to anneal to the gene at particular spot.
PCR is done by mixing chromosomal or plasmid DNA with a master mix and initiated by
adding Taq polymerase (Nelson et. al. 2008). The tubes are put into a thermal cycler that heats
and cools the tubes following a thermal program for each cycle. For each cycle there is a
doubling of DNA corresponding to the region subtended by the primers (Raven 2013). A typical
run consists of 30 cycles, resulting in an exponential amplification. For example, if the tube had
only one copy of the gene being amplified, after 30 cycles there would be 5 x 10
8
copies (Raven
2013).
After isolation and amplification comes separation and analysis. The PCR products will
be examined with restriction enzyme digestion and separated on agarose gel electrophoresis. Gel
electrophoresis is used in this experiment to separate a population of DNA fragments by length
(Kryndushkin et. al. 2003). Ethidium bromide, fluorescent when incorporated into dsDNA, is
used as an indicator of the presence of DNA. The gels will be viewed with ultraviolet light to
further stimulate ethidiuim bromide fluorescence (Kryndushkin et. al. 2003).
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Methods

DNA isolation from liver
1. A 1.5 ml centrifuge tube was weighed.
2. A 25 mg piece of liver was cut and inserted into the centrifuge tube.
3. The centrifuge tube with the sample was weighed again and the tissue weight was recorded.
4. Sample was mashed with a plastic pestle.
5. 180 l of Lysis Solution T was added.
6. 20 l of Proteinase K solution (0.25ml water to stock bottle) was added.
7. The sample was mixed immediately mix via vortexing and placed into the 55
o
C water bath.
8. The sample was checked every 5 min, and vortexed until the tissue sample becomes
homogeneous.
9. 20 l of RNase A solution was added and the sample was incubated for 2 mins at room
temperature.
10. 200 l of Lysis Solution C was added, sample was vortexed, and placed in the 70
o
C water
bath for 10 minutes.

Spin Column Prep
11. 500 l of Column Preparation Solution was added to a spin column. The sample was
centrifuged at 12,000 x g for 1 minute.

DNA Separation
12. 200 l of ethanol was added to the tissue lysate and the sample was vortexed for 10-20 sec
until homogenous.
13. The contents of the tissue lysate were transferred to the spin-column tube using a wide bore
pipet to avoid shearing DNA.
14. Sample was centrifuged at >6500 x g for 1 minute and tube was discarded the tube.
15. The spin column was placed in a fresh tube, 500 l of Wash Solution (previously prepared
with ethanol: add 10 ml ethanol to Wash Solution Concentrate bottle) was added to tube.
16. The sample was centrifuged at >6,500 x g for 1 minute and the collection tube was discarded
and the spin column was placed in a new tube.
17. 500 l of Wash Solution was added and the sample was centrifuged for 3 minutes at
maximum centrifuge speed. This was repeated as necessary to remove ethanol from the column.
18. DNA was eluted by pipetting in 200 l of Elution Solution directly into the center of the spin
column.
19. Sample was incubated for 5 minutes at room temperature, then centrifuged at >6,500 x g for
1 minute. (The sample is to be stored short-term stored at 2-8oC or frozen for long term storage.)

UV Spectrophotometry
20. Two 1 ml cuvettes with TE buffer. (TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0-8.5)
were matched at 260nm to a pre-warmed up UV-Vis spectrophotometer.
21. 10 l of DNA preparation was added to sample cuvette and the absorbance was found from
200-400 nm.

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PCR Procedure.
22. 30 l of master mix was placed into a PCR tube.
23. Approximately 20 l or 100ng DNA sample was added. The values were calculated from the
UV data and the difference was made up with reagent grade water if the volume was less than 20
l. (1.0 absorbency at 260 nm of double stranded DNA corresponds to 50 g DNA/ml.)
24. The sample was heated to 95
o
C for 3 minutes.
25. PCR was run for 30 cycles of 95
o
C for 1 min => 52oC for 1 min => 72oC for 1 min.
26. For the last cycle the sample remained at 72
o
C for 10 min.
27. The sample was chilled to 4oC over night.

Agarose Gel Electrophoresis
28. The edges of a clean, dry glass plate (or the open ends of the plastic tray supplied with the
electrophoresis apparatus) were sealed with tape to form a mold. The mold was set on a
horizontal section of the bench.
29. Sufficient electrophoresis buffer (usually 1x TAE or 0.5x TBE) to fill the electrophoresis
tank and to cast the gel was prepared. The same batch of electrophoresis buffer was used in both
the electrophoresis tank and the gel.
30. A solution of agarose was prepared in electrophoresis buffer at a concentration appropriate
for separating the particular size fragments expected in the DNA sample: The correct amount of
powdered agarose (please see table below) was added to a measured quantity of electrophoresis
buffer in an Erlenmeyer flask or a glass bottle.

Range of Separation in Cells Containing Different
Amounts of Standard Low-EEO Agarose
Agarose Concentration
in Gel (% [w/v])
Range of Separation of
Linear DNA Molecules (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0.2-3
2.0 0.1-2
31. The agarose was melted in the presence of the desired buffer until a clear, transparent
solution was achieved. The melted solution was poured into a mold and allowed to harden.
32. The slurry was heated in a microwave oven until the agarose dissolves.
33. Insulated gloves or tongs were used to transfer the flask and ethidium bromide was added to
a final concentration of 0.5g/ml. The gel solution was mixed by gentle swirling.
34. The warm agarose solution was poured into the mold.
35. Once the agar has cooled slightly, a suitable comb was inserted into the mold.
36. The gel was allowed to set completely for 30 minutes and the comb was removed.
37. The gel was mounted in the electrophoresis tank and sufficient electrophoresis buffer was
added to cover the gel completely.
38. The samples of DNA were mixed with 0.20 volumes of the desired 6x gel-loading buffer.
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39. The DNA samples and the DNA standard were loaded into the slots in the submerged gel
using a disposable micropipette.
40. The lid of the gel tank was closed and the electrical leads were added so that the DNA will
migrate toward the positive anode (red lead).
41. A voltage of 1-5 V/cm (measured as the distance between the positive and negative
electrodes) was applied and the electrophoresis was allowed to run for 45 minutes.
42. The gel tray was examined by UV illumination on a trans-illuminator and observations were
recorded.

Results

The gel showed one band from each well totaling five bands.

Discussion

The purpose of this experiment, to isolate and characterize DNA from a biological
sample and amplify the cytochrome-b gene, was accomplished. The DNA sequence for
cytochrome b from Gallus gallus is:
acacagcaga cacttcccta gccttctcct ccgtagccca cacttgccgg aacgtacaat
acggctgact catccggaat ctccacgcaa acggcgcctc attcttcttc atctgtatct
tccttcacat cggacgaggc ctatactacg gctcctacct ctacaaggaa acctgaaaca
caggagtaat cctcctcctc acactcatag ccaccgcctt tgtgggctat gttctcccat
ggggccaaat atcattctga ggggccaccg ttatcacaaa cctattctca gcaattccct
acattggaca caccctagta gagtgaggct gagggggatt ttcagtcgac aacccaaccc
ttaccggatt tttcgcctta cacttcctcc tcccctttgc aatcgcaggt attactatca
tccacctcac cttcctacac gaatcaggct caaacaaccc cctaggcatc tcatccgact
ctgacaaaat tccatttcac ccatactact ccttcaaaga cattctgggc ttaactctca
tactcacccc attcctaaca ctagccctat tctcccccaa cctcctagga gacccagaaa
acttcacccc agcaaaccca ctagtaaccc cccc (Esposti et. al.1993)
The electrophoresis gel showed one band for each sample, totaling five distinct bands.
This suggested that the restriction enzyme was successful in isolating its respective portion of the
sample. In the event of multiple bands being visible, this would suggest that the restriction
enzyme stuck to more than one place on the sample. Possible sources of error for this experiment
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include: unknown errors accumulated during the PCR process completed by the TA, losing
product during the process of acquiring crude cellular material from the liver tissue because not
all of the solution was collected from the mortar and pestle, and because the yield was ~2 g/L
the TA added extra template to reach a concentration of 50g/L possibly altering the bands
seen in the gel electrophoresis.
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Works Cited
Esposti MD, De Vries S, Crimi M, Ghelli A, Patarnello T, Meyer A (July 1993). "Mitochondrial
cytochrome b: evolution and structure of the protein". Biochim. Biophys. Acta 1143 (3):
24371. Print.
Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, Kushnirov VV (2003). "Yeast [PSI+]
prion aggregates are formed by small Sup35 polymers fragmented by Hsp104". Journal
of Biological Chemistry 278 (49): 4963643. Print.
Nelson, David L. et. al. Principles of Biochemistry. W. H. Freeman and Company: New York,
Print. 2008.
Raven, Peter, et. al. Biology. McGraw-Hill. Print. 2013.