A.3.2.3BreastCancerElectrophoresis



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|Activity 3.2.3: Breast Cancer Screening and Prevention with Electrophoresis (Optional) |

Introduction

Judy Smith leads a busy life balancing her work as a dental hygienist at a local dentist office and being a mother of three. Although her life is very hectic, she is happy and relatively stress free. She is slightly overweight but walks at least two miles every day. Judy’s mother, Laura, and maternal grandfather, Bill, were both diagnosed with breast cancer when they were in their forties. Judy’s sister, Jennifer, was diagnosed with breast cancer when she was 35. After her son was diagnosed with osteosarcoma, Judy started thinking more about her own health. Now that she is in her forties, she worries that she is at greater risk for developing breast cancer because of her family history of the disease. Judy’s husband, James, suggested that she should have genetic testing done to find out if she inherited the altered genes known to cause breast cancer.

The two genes associated with inherited cases of breast cancer are called BRCA1 (breast cancer gene 1) and BRCA2 (breast cancer gene 2). When functioning normally, these two genes act as tumor suppressor genes. This means that they help repair DNA damage and prevent cancer cell formation. If these genes become mutated, this can lead to the development of breast or ovarian cancer. About five to ten percent of all breast cancers are associated with BRCA1 and BRCA2 mutations. It is estimated that an average woman in the United States has a 12% chance of developing breast cancer in her lifetime. This risk increases to up to 85% if the woman has a mutated BRCA1 or BRCA2 gene.

Judy Smith decides to find out if she has abnormal BRCA1 or BRCA2 genes. In this activity, you will perform marker analysis using gel electrophoresis to analyze Judy’s DNA, as well as the DNA of her family members, and assess their risk for breast cancer. After you have obtained Judy’s test results, you will make recommendations for Judy on what she can do to lower her risk.

Equipment

• Computer with Internet access

• Laboratory journal

• Activity 2.1.1 Student Response Sheet

• Activity 3.2.3 Student Response Sheet

• WARD’S Detection of Hereditary Breast Cancer Lab Kit

o DNA Samples: PCR Samples for four patients, Size Markers

o Agarose

o TBE Running Buffer

o Gel Staining Equipment and Reagents

• Micropipettors and tips

• Electrophoresis chamber and power supply

• UV transilluminator or White light box

• Ruler

Procedure

Part I: The Genetics of Breast Cancer

1. Watch the video clip A Family Disease taken from NOVA’s Cracking the Code of Life program, found at: .

o Click on video clip #14, A Family Disease.

2. Reflect on what you watched in the video. In Activity 2.1.1, you took a genetic testing survey. Now that you know more about genetic testing, do you feel the same way?

3. Take out Activity 2.1.1: Student Response Sheet. Read through your original answers.

4. In your laboratory journal, write a reflection on whether or not you have changed your mind about any of your answers.

o If you had a chance of having a mutation in either your BRCA1 or BRCA2 gene, would you get tested? Explain your answer in your laboratory journal.

o How is testing for BRCA1 or BRCA2 different from testing for a gene such as the Tay-Sachs gene? Explain your answer in your laboratory journal.

Most cases of breast cancer are not due to BRCA1 and BRCA2 mutations. Genetic testing can be very expensive; before testing is done, a pedigree is constructed and the family’s breast and ovarian cancer pattern is analyzed. Cancers can be divided into three categories: sporadic, familial, and hereditary.

5. Go to the GeneticHealth’s website and read the article Hereditary Versus Spontaneous Cancer, found at:

6. Take notes on the differences between sporadic, hereditary, and familial cancers in your laboratory journal.

7. Add the information provided about Judy’s family breast cancer occurrence given in the Introduction of this activity to your Smith family tree.

8. Use the notes in your laboratory journal, as well as the information found in the following website, and decide if you believe Judy is likely to have an inherited BRCA1 or BRCA2 mutation.

o ’s “Assessing Your Genetic Risk” found at:

9. Answer Conclusion questions 1 and 2.

Judy’s doctor believes that the cases of breast cancer in Judy’s family are consistent with hereditary cancer. Because both males and females are affected, and because there are no cases of ovarian cancer, the doctor suspects a mutation in the BRCA2 gene. The BRCA2 gene contains more than 80,000 nucleotides and is larger than the average gene. Researchers have identified more than 600 mutations in the BRCA2 gene, many of which are associated with an increased risk of breast cancer. Many BRCA2 mutations insert or delete a small number of nucleotides in the gene. Because the BRCA2 gene is a tumor suppressor gene, the mutation will result in a protein that is unable to help repair damaged DNA or fix mutations.

Because Judy’s sister, Jennifer, developed breast cancer at an early age, she is the most likely member of this family to have a BRCA2 mutation. Therefore, she is the best candidate for genetic testing. Jennifer agrees to be tested, and undergoes DNA sequencing of her BRCA1 and BRCA2 genes. Jennifer tests negative for a BRCA1 mutation and tests positive for a genetic mutation of the BRCA2 gene known to be associated with breast cancer. Judy’s mother, Laura, also undergoes DNA sequencing, and tests positive for the same genetic mutation of the BRCA2 gene as Jennifer. Armed with this knowledge, Judy proceeds with getting tested and also convinces her sister Diana to be tested. However, DNA sequencing is extremely expensive. Judy’s doctor recommends a type of genetic testing called marker analysis which is less expensive and takes less time than DNA sequencing.

Marker Analysis is a technique where the gene mutation is analyzed using a genetic marker instead of directly analyzing the gene itself. A genetic marker is a short sequence of DNA associated with a particular gene or trait with a known location on a chromosome. The genetic markers used in marker analysis are short DNA sequences called Short Tandem Repeats (abbreviated STRs and also called microsatellites). An STR is a region of DNA composed of a short sequence of nucleotides repeated many times. The number of repeated sequences in a given STR varies from person to person. The alternate forms of a given STR correspond with different alleles. Most STRs occur in gene introns (non-coding regions of DNA), so the variation in the number of repeats does not usually affect gene function, but we can use STRs to differentiate between different alleles. Because pieces of DNA that are near each other on a chromosome tend to be inherited together, an STR that is located on chromosome 13 next to the known BRCA2 mutation can be used as the genetic marker for this case. The diagram below shows the relationship between the gene of interest and the genetic marker:

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In order to test Judy and her family members for the BRCA2 mutation, DNA is extracted from each family member. The region of DNA containing the STR which is going to be used as the genetic marker for this mutation is amplified using PCR. The amplified DNA will then be run on a gel using gel electrophoresis. Because different alleles have a different number of repeats present in the STR, gel electrophoresis will separate different alleles based on the number of repeats present. The more repeats present in an STR, the longer the DNA fragment will be. The shorter DNA fragments will migrate the farthest down the gel.

You have been assigned to perform marker analysis for the Smith family. You will be provided with PCR products for this marker produced from DNA specimens from Jennifer, Judy, Laura, and Diana. Your job is to run a gel electrophoresis with your partner(s) to determine who has the BRCA2 mutation. You will be able to identify the different alleles by determining the band lengths for each family member. Because each person has two chromosome 13’s, each person should have two alleles for this marker. You will then have to identify which allele is linked to the mutant gene by determining which alleles Jennifer and Laura have in common (since they are both known to have the mutation), and see if this allele is present in Diana and Judy.

10. Answer Conclusion question 3.

The pedigree below shows the relationship between the family members you will be testing. Remember: Bill, Laura, and Jennifer are all filled-in because they were diagnosed with breast cancer.

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Part II: Gel Electrophoresis

11. Obtain a solidified agarose gel, an electrophoresis chamber, and a power supply from your teacher.

12. Place the solidified gel in the electrophoresis chamber and add enough 1X TBE buffer to cover the surface of the gel.

13. Carefully remove the comb. Make sure that the wells are submerged in buffer. If necessary, add additional TBE buffer.

14. Using a fresh tip for each sample, load 10 μL from each sample tube onto the corresponding gel lane with a micropipettor. Be careful to not pierce the bottom of the wells with the micropipette tip and do not overload the wells.

Lane #1: DNA Size Markers

Lane #2: Diana’s DNA

Lane #3: Jennifer’s DNA

Lane #4: Laura’s DNA

Lane #5: Judy’s DNA

Lane #6: Negative Control

15. Answer Conclusion question 4.

16. Draw the following diagram in your laboratory journal. Make sure to label which samples were loaded into each well.

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17. Run the gel at 130V for 30 minutes or until the dye front has moved almost to the end of the gel.

18. Stain the gel according to your teacher’s instructions. This part may be completed for you.

19. View the gel using a white light box or a UV transilluminator.

Part III: Analysis – Standard Curve Plot

Remember that the basic principle behind DNA gel electrophoresis is that negatively charged DNA migrates through the porous lattice in the agarose gel. Larger DNA fragments migrate slower through the lattice than the smaller fragments, since the smaller fragments can fit through the holes easier. Therefore, when run through an agarose gel, the DNA fragments are separated by size. You loaded DNA size markers in the first well of your gel. These DNA size markers are a set of DNA fragments of known molecular sizes. They can be used as a standard to determine the sizes of your unknown fragments. Your results should be similar to the diagram below. The known molecular sizes (weights in base pairs) for each marker are written to the left of each band.

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By measuring the distance migrated from each of the standard markers, you can construct a standard curve. However, the distance a DNA fragment migrates is not a linear function of its size. A molecule ten times the size of another molecule will only migrate half as far and not 1/10th as far. Therefore, to construct a standard curve, you need to plot the relative mobility value (Rf) for each standard marker versus their molecular size in base pairs on a semi-logarithmic graph. You will first calculate the Rf for each standard marker using the following formula:

Distance the DNA fragment has migrated from the origin (gel well)

Rf = Distance from the origin (gel well) to the reference point (tracking dye)

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Once you create the standard curve, you will then use the standard curve to determine the molecular size of the DNA fragments from each family member being tested.

20. Measure the distance in mm from the sample well to each fragment in the standard lane. Record each in the Table One, in order, beginning with the band closest to the sample well under Distance Migrated (mm).

21. Measure the distance from a sample well to the end of the gel. Record the number in Table One under Distance to Reference Point. This number will be the same for each size marker fragment.

22. Calculate the Rf of each fragment and record in Table One. Round values to the nearest one hundredth.

Table One:

|DNA Size Markers |Fragment Length in Base |Distance Migrated (mm) |Distance to Reference Point |Rf |

| |Pairs |A |(mm) | |

| | | |B |A ÷ B |

|Fragment 1 |1353 | | | |

|Fragment 2 |1078 | | | |

|Fragment 3 |872 | | | |

|Fragment 4 |603 | | | |

|Fragment 5 |310 | | | |

|Fragment 6 |281 | | | |

|Fragment 7 |234 | | | |

|Fragment 8 |194 | | | |

23. Obtain a Student Response Sheet from your teacher.

24. Draw a standard curve plotting Rf versus fragment length (in base pairs) on your Student Response Sheet.

25. Calculate the Rf value for each DNA fragment for each family member and fill-in the table below. Round values to the nearest one hundredth.

|DNA Sample: |Fragment: |Distance Migrated (mm) |Distance to Reference |Rf |

| | |A |Point (mm) | |

| | | |B |A ÷ B |

|Diana |Fragment 1 | | | |

| |Fragment 2 | | | |

|Jennifer |Fragment 1 | | | |

| |Fragment 2 | | | |

|Laura |Fragment 1 | | | |

| |Fragment 2 | | | |

|Judy |Fragment 1 | | | |

| |Fragment 2 | | | |

26. Use your standard curve to determine the base pair size of the fragments of all family members’ DNA samples and add this information to Table Two.

Table Two:

| DNA Sample: |Fragment: |Fragment Length (in base pairs) |Allele Present: |

|Diana |Fragment 1 | | |

| |Fragment 2 | | |

|Jennifer |Fragment 1 | | |

| |Fragment 2 | | |

|Laura |Fragment 1 | | |

| |Fragment 2 | | |

|Judy |Fragment 1 | | |

| |Fragment 2 | | |

27. Use the data table below to determine which alleles were present in each family member’s DNA samples. Fill in the Allele Present column in Table Two.

| Fragment Length in Base Pairs: |Allele: |

|200 |Allele 1 |

|300 |Allele 2 |

|400 |Allele 3 |

|500 |Allele 4 |

|600 |Allele 5 |

|700 |Allele 6 |

|800 |Allele 7 |

|900 |Allele 8 |

|1000 |Allele 9 |

28. Answer Conclusion questions 5 - 7.

Part IV: Prevention

Women who have an abnormal BRCA1 or BRCA2 gene have a 50-80% risk of breast cancer by the age of 70. Their lifetime risk for ovarian cancer is also increased. Even if you do not have an increased risk for developing breast cancer, there are many lifestyle choices persons can make to reduce their risk. Women who have an increased risk due to an abnormal gene also have other preventative options to reduce their risk, such as medication and/or surgery.

29. Research the preventative measures that can be taken to reduce the risk of developing breast cancer. Use the sites listed below, as well as other reliable Internet resources you might find.

o American Cancer Society’s The Complete Guide – Nutrition and Physical Activity, found at:

o ’s Hormonal or Anti-estrogen Therapy, found at:

o ’s Prophylactic Mastectomy, found at: Preventative Surgery Options, found at:

o National Academy of Sciences’ Exercise May Reduce Risk of Breast Cancer, found at:

30. Include information on the following preventative measures in your notes:

o Nutrition

o Physical Activity

o Hormonal or Anti-estrogen Therapy

o Prophylactic Surgeries

31. Write notes from your research in your laboratory journal. Make sure to properly cite your sources.

32. Write a letter to Judy Smith from the perspective of her doctor. In the letter, explain your findings from her genetic test and what the results mean. Explain to Judy what preventative measures she can take to reduce her risk for developing breast cancer and what options she has. Use your notes from your laboratory journal to compose your letter.

33. Answer the remaining Conclusion questions.

Conclusion

1. Explain whether you think Judy’s family occurrences of breast and ovarian cancers are sporadic, hereditary, or familial.

2. Is Judy a good candidate for BRCA1 or BRCA2 genetic testing? Explain your answer.

3. Draw a diagram showing how marker analysis works.

4. Why did you use a negative control when running gel electrophoresis? If a band shows up in this lane, what does it tell you about your PCR products?

5. Which allele is associated with the BRCA2 mutation? Explain your answer.

6. Which family members have the BRCA2 mutation? Explain your answer.

7. Do you think this type of genetic testing should be available? What are the advantages and disadvantages of knowing what is in your genes?

8. How do you feel about prophylactic surgeries for those who test positive for the breast cancer gene? Explain.

34. The lifetime chance of developing breast cancer is greatly increased in someone who has inherited an altered BRCA1 or BRCA2 gene. What role does environmental risk play into whether or not this person actually develops breast cancer?

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