Cancer Gene

Detection/Restriction
Enzyme Mapping

By Grant Akalonu
CHE 447-01
Objective/Introduction

The purpose of this experiment was to study the detection of cancer genes, and to
understand how restriction enzymes can be used in forensic DNA fingerprinting. In this
experiment, cancer formation based on alteration of the gene for p53 tumor suppressor protein.
The gene for the p53 protein is located on the short arm of chromosome 17. Normally, the p53
gene is a sequence specific DNA-binding protein which acts as a transcriptional regulator. When
this gene is altered by mutations, it loses its ability to bind to DNA. Also, p53 can develop
mutations in specific hot spots which promote uncontrolled cell growth. A simulation will be
performed to assess the risk of Li-Fraumeni syndrome for a woman names Valerie. This is done
by digesting her DNA with a restriction enzyme and separating the fragments using agarose gel
electrophoresis, and constructing a family pedigree.
Restriction enzyme mapping involves the activity of restriction endonucleases, a class of
enzymes that cleave double stranded DNA at specific locations. The likelihood that a restriction
enzyme cleaves a portion of DNA is proportional to the length of its recognition site. Restriction
enzymes can be used for DNA fingerprinting by exposing two different DNA samples to the
same restriction enzyme and comparing the fragments of DNA that the enzyme creates. In this
experiment DNA samples from two suspects in a crime scene simulation are cleaved with the
same restriction enzyme to see if there is a match between any of the suspects and the crime
scene.

Materials & Methods

Cancer Gene Detection

50X TAE electrophoresis buffer was diluted to 1X.

0.8% agarose gel was prepared by mixing 0.23 g of UltraSpec agarose powder in 30mL
of 1X TAE electrophoresis buffer. The mixture was heated in a microwave and then
cooled to 60C before being poured into the bed with the comb sitting firmly and evenly
across the bed.
16 L of each sample was loaded into the gel and ran a 135V for an hour. Then the gel
was stained with ethidium bromide and visualized under UV light.
The wells of a microtiter plater were labeled as follows:
(-) negative control
(+) positive control
(P1) patient 1
(P2) patient 2
(P3) patient 3
(CAP) used to add/remove the capture antibody
(PBS) used to add PBS buffer to each well
(SUB) used to add substrate to each well
(DET) used to add detection antibody to each well
50 L of the capture antibody solution was added to all of the wells. The plate was then
incubated at room temperature for 5 minutes.
 The capture antibody solution was removed form the wells using the proper pipet. Each
well was then washed with PBS buffer.
50 L of the patients samples was added to the wells. The samples were the nincubated
at 37C for 15 minutes.
The patients samples were removed using the appropriate pipet, and then washed with
PBS buffer.

Restriction Enzyme Mapping

50X TAE electrophoresis buffer was diluted to 1X.

0.8% agarose gel was prepared by mixing 0.23 g of UltraSpec agarose powder in 30mL
of 1X TAE electrophoresis buffer. The mixture was heated in a microwave and then
cooled to 60C before being poured into the bed with the comb sitting firmly and evenly
across the bed.
DNA 1, DNA 2, Enzyme 1, Enzyme 2 were obtained from the instructor.
The enzymes were added to the suspects DNA as shown in Figure 1.

Figure 1. Summary of restriction enzyme digestion reactions

The tubes were then incubated at 45C for 15 minutes.

The standard maker, CS1, and CS2 were obtained from the instructor.
After the incubation, 5 L of 10X loading solution was added to the stop the reaction.
The 6 well gel was used, and 35 L of each sample was loaded into to the gel.
The gel was put to run at 135V for 1 hour, and then stained with ethidium bromide for 10
minutes before visualization over UV light.
Results
Lane Sample
1 Standard DNA
Maker
2 Control DNA
3 Patient Peripheral
Blood DNA
4 Patient Breast
Tumor DNA
5 Patient Normal
Breast Tissue DNA

Fig.1. Cancer Agarose Gel Result

Fig.2. ELISA C-Peptide Result

Fig.3. Family Pedigree

 Well Sample
1 Crime scene DNA cleaved
with enzyme 1
2 Crime scene DNA cleaved
with enzyme 2
3 Suspect 1 DNA cleaved
with enzyme 1
4 Suspect 1 DNA cleaved
with enzyme 2
5 Suspect 2 DNA cleaved
with enzyme 1
6 Suspect 2 DNA cleaved
with enzyme 2
Fig.4. Restriction Enzyme Gel Result.

Discussion & Conclusion

Based on the results, it appears that there was a malfunction with the agarose gel for the
cancer gene detection experiment. The DNA marker is barely visible, however some infromatin
can still be drawn. There is only one band for the normal control DNA, which means that the
restriction enzyme did not cleave the DNA segment because there is not restriction enzyme site.
Therefore, it will appear as only one band. For the patient DNA with the tumor, there is two
bands, indication that the restriction enzyme was able to cleave the DNA into fragments.

For the restriction enzyme mapping, it appears that suspect 2s DNA is closest to that of
the crime scene. The pattern which the enzymes cleave the DNA at the crime scene shown in
wells 1 and 2 is the same pattern observed when both enzyme clave suspect 2s DNA. Therefore,
this experiment was able to successfully use restriction enzymes to perform forensic DNA
fingerprinting.