Name:                                                                 Block:

In Search of Cancer Gene Lab[1]

Many contributory factors have been identified to cause the onset of cancer that include exposure to certain carcinogens (toxins) in our diets and environment. Several forms of cancer can be based on inheritance from family members. These cancers appear to be linked to inherited mutation of genes, such as p53.

                                                                           

With the advent of molecular biology applications to medicine, gene maps and the chromosomal locations of genes are available as tools for the identification of predisposition for various diseases. The procedures used to obtain such information include DNA isolation and the analysis of mutations in hotspot areas in cancer-related genes, such as p53. Several methods of analysis for the detection of mutations in genes include DNA sequencing (separating DNA to look for mistakes or errors).

                                                                           

The Human Genome Project has provided information to link identification of various cancers and other diseases to DNA sequencing information.  The study of inherited cancers has given cancer molecular biologists the opportunity to search for genes that are critical in normal cell development and cancer. At the molecular level, cancer formation is characterized by changes in genes, such as p53.  Cancer is most simply caused by uncontrolled cell growth and division.

                

In recent years, the p53 tumor suppressor gene has become the center of many cancer biology studies. Because it appears to be of major significance, there is great importance to study how this gene functions in normal cells compared to cancer cells. The gene for the p53 protein is located on the short arm of chromosome 17. The gene p53 usually acts as a cell growth regulator (stops cells from continually dividing).  Evidence suggests that mutations in the p53 gene promotes uncontrolled cell growth and potentially cancer.  Mutations to the p53 gene is generally called Li-Fraumeni syndrome.  This rare disorder affects young family members and results in high death rates as caused by cancer.  This disorder can be passed from parents to offspring.

                                                                

Part A: Constructing a Family Pedigree

A first step in the search and assignment of Li-Fraumeni syndrome is to establish the family pedigree of the patient.

                        

The first part of the experiment is based on the information made available as part of a diagnosis by the family physician and the oncologist. The pedigree information that you will develop is for a young woman who is suspected to have the Li-Fraumeni syndrome.

                        

INFORMATION FOR DEVELOPING THE FAMILY PEDIGREE:                        

Upon monthly breast self-examination, Valerie Brown, age 36, found a small irregular mass. She was concerned because she knew that her mother had a mastectomy (breast removal) when she was in her late thirties. Valerie made an appointment with her physician, who referred her to a specialist at a local cancer center, where she was diagnosed as having breast cancer. As part of the medical work-up, the oncologist had inquired about her family history of cancer. Upon consultation with her mother, Valerie learned that her father and his family appeared to be free of cancer. However, in Valerie's mother's family, several cases of cancer have occurred.

With the information given below, chart the family pedigree.

All the children show no signs of cancer at this time.  Valerie has requested that DNA sequencing be done for each of her children.

CREATE THE FAMILY PEDIGREE BELOW


Part B: Separation of DNA Fragments

The family pedigree in Module 1 strongly suggests Li-Fraumeni syndrome. In such a case, a secondary diagnostic test is normally conducted. In this scenario, Valerie provides a sample of blood and tumor biopsy tissue to conduct DNA analysis for the p53 gene. Normally the procedure is to amplify the gene using polymerase chain reaction (process that makes millions of copies of a single portion of the patient’s DNA). This is followed by one of several methods to detect the presence of a point mutation at the hot spots.

In this simulation experiment, Valerie's DNA has already been digested with a restriction enzyme that recognizes the mutant sequence at the simulated hot spot site at nucleotide 165 which is also the palindrome CAGCTG for the restriction enzyme. This restriction enzyme was used as a probe to cut the simulated amplified gene for Valerie’s DNA sample, together with a normal control and a set of standard DNA marker fragments. Digestion of the normal amplified DNA will give a characteristic "control" DNA fragment banding pattern. The DNA obtained from blood lymphocytes will give an altered band pattern representing one normal allele and the second which is the mutant. The DNA analysis from the tumor tissue will show only the pattern for the tumor allele. The pre-digested DNA samples with the control wild type and DNA markers will be separated by agarose gel electrophoresis, stained, and then analyzed.

Agarose Gel Requirements:

Preparing the Agarose Gel        

  1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams
  2. Place a well-former template (comb) in the first set of notches at the end of the bed. Make sure the comb sits firmly and evenly across the bed.
  3. Place the bed on a level surface and pour the cooled agarose solution that your teacher has prepared into the bed.
  4. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 15 minutes; while you are waiting your teacher may have you begin additional steps.
  5. After the gel is solidified, be careful not to damage or tear the wells while removing the rubber dams and comb from the gel bed.
  6. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.
  7. Fill the electrophoresis apparatus chamber with diluted (1x) electrophoresis buffer so that the gel is covered by at least approximately 5cm.                                 

                                         

Loading DNA Samples & Running the GEL                

  1. Heat the DNA fragments (tubes A-E) for two minutes at 65°C. Allow samples to cool for a few minutes before gel loading.
  2. Load 18 μl of each DNA sample in the following manner:

                

Lane

Tube

Sample Tested

1

A

Standard DNA Fragments

2

B

Control DNA

3

C

Patient Peripheral Blood DNA

4

D

Patient Tumor DNA

5

E

Patient Breast Normal DNA

  1. After the DNA samples are loaded, properly orient  the cover  (colors match-up) and carefully snap it onto the electrode terminals.
  2. Insert the plugs of the black and red wires into the corresponding inputs of the power source.
  3. Plug in the power cord to the electrophoresis unit; have your teacher check before proceeding.
  4. Set the power source at 125 volts and start a timer for 25 minutes
  5. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes.
  6. After the electrophoresis is completed, turn off the electrophoresis apparatus, disconnect the electrophoresis power cord from the outlet, and disconnect the power and remove the gel from the bed for staining.

Staining & Visualization of DNA                                                                           

  1. After electrophoresis, place the gel on a piece of plastic wrap on a flat surface. Moisten the gel with a few drops of electrophoresis buffer.
  2. Wearing gloves and safety goggles, remove the clear plastic protective sheet, and place the unprinted side of the InstaStain® EtBr card on the gel.
  3. Firmly run your fingers over the entire surface of the InstaStain® EtBr. Do this several times. Wear gloves and safety goggles
  4. Place the gel casting tray and a small empty beaker on top to ensure that the InstaStain® card maintains direct contact with the gel surface.  Allow the InstaStain® EtBr card to stain the gel for 15 minutes.
  5. After 15 minutes, remove the InstaStain® EtBr card. Transfer the gel to a ultraviolet (300 nm) transilluminator for viewing. Be sure to wear UV protective goggles.

                                        

                                

Gel Analysis

Your group has conducted analysis for one of Valerie’s children.  You will share your data with the entire class for analysis. In this part of the experiment, x-ray results of the wild p53 and samples from Valerie's five children will be read to determine whether or not there are mutations.

Valerie’s Children

Ages

Gel #

Justin

16

1

Shelia

14

2

Robert

10

3

Angela

8

4

Anthony

6

5

                                                                 

                                

                                         

Analysis:

For each of Valerie's children, obtain data for each of Valerie’s Children from the table above.  The sequencing reactions have all been loaded in order: G-A-T-C.  Begin analysis of the DNA sequence at the bottom of the gel with the circled band, which is an A.  Compare the deduced sequence to the wild type sequence shown in the box below.

  1. Based on the information obtained from the Gels, which of Valerie’s children have a mutation in their DNA sequence and how do you know?
  1. Justin:

  1. Shelia:

  1. Robert:

  1. Angela:

  1. Anthony:

  1. What is the difference between tumor suppressors and oncogenes?         

  1. Why does Valerie’s tumor DNA sample have fewer bands than the peripheral blood?

  1. What is the purpose of the control lane?

  1. Could a doctor proceed with diagnosing Valerie’s condition based on the molecular biology data from this lab?  Explain why or why not.        

                                         

                

                

Assessment:

Please see your teacher’s website for explanation of proficiency level for each standard

Science Standard 1: Planning & Evaluation

Exceeds Proficiency

Meets Proficiency

Nearly Proficient

Not Proficient


[1] Adapted from Edvo Kit #314: In Search of the Cancer Gene