1. DNA marker analysis Mrs. Stewart Medical Interventions Central Magnet School

2. Standard: Marker analysis is a technique used to determine the presence of genetic mutations associated with cancer. Objective: Investigate the marker analysis technique and analyze results to determine the presence of a BRCA 2 mutation

3. 3 Cancer Categories Sporadic Hereditary Familial

4. Judy Smith 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.

5. BRCA 1 and BRCA 2 Tumor suppressor genes Repair DNA damage and control cell growth Proto-oncogenes

6. BRCA 2 80,000 nucleotides (larger than average gene) 600+ mutations identified and linked to increased risk of cancer Most mutations are insertion or deletion mutations (frameshift) of more than one base Results in a protein that is unable to help repair damaged DNA or fix mutations

7. Judy Smith 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.

8. Family Pedigree Draw a pedigree for the Smith family to represent the mutated BRCA2 gene.

9. Marker Analysis Technique A technique where a gene mutation is analyzed by using a genetic marker instead of directly analyzing the gene itself. Genetic marker: Alteration in DNA that may indicate an increased risk of developing a specific disease or disorder

10. STRs Genetic markers used in marker analysis are short DNA sequences called Short Tandem Repeats (STRs) STRs – region of DNA composed of a short sequence of nucleotides repeated many times. # of repeated sequences vary Alternate forms of STRs correspond with different alleles.

11. STRs Most STRs occur in gene introns (non-coding regions of DNA) Does not usually affect gene function Can use as “markers” to differentiate between different alleles for certain genes (because genes located next to each other are inherited together.)

12. PCR The region of the DNA that is the known STR marker is amplified using PCR (and the BRCA unknown gene version with it) The amplified DNA is then run on a gel.

13. 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 = the longer the DNA fragment Shorter DNA fragments migrate farther down the gel So, the fragments that migrate the farthest, have the fewest STRs

14. Standard: Marker analysis is a technique used to determine the presence of genetic mutations associated with cancer. Objective: Investigate the marker analysis technique and analyze results to determine the presence of a BRCA 2 mutation

15. Mission Objective 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.

16. Smith Family Analysis We will determine different alleles for each family member tested by determining the band lengths for each family member. Who was tested? Diana –Judy’s sister Jennifer – Judy’s sister Laura – Judy’s mom Judy – Sue, Mike and Tucker’s mom

17. Who has the BRCA 2 mutation? Each person has 2 chromosomes #13, so each person will have 2 alleles for the BRCA 2 gene. You will have to identify which allele is linked to the mutated version of the gene by determining which alleles Jennifer and Laura have in common Since both of them are known to carry that allele. Analyze Judy and Diana’s results to determine if they also carry the mutated gene allele.

18. Analysis DNA size markers are loaded in the first well. Use the size markers to determine the sizes of your unknown fragments. The known molecular size markers (weights in base pairs) are written beside each band. Measure the distance each unknown band migrated

19. Data Distance Migrated (mm) - Measure the distance in mm from the sample well to each fragment in the standard lane. Record in Table One Distance to Reference Point - Measure the distance from the sample well to the end of the gel. Record in Table One. This number will be the same for each size marker fragment. Calculate the R f of each fragment and record in Table One. Round values to the nearest one hundredth. DNA Size Markers Fragment Length in Base Pairs Distance Migrated (mm) A Distance to Reference Point (mm) B R f A ÷ B Fragment 11353 Fragment 21078 Fragment 3872 Fragment 4603 Fragment 5310 Fragment 6281 Fragment 7234 Fragment 8194 Table One:

20. Plot your standard curve Plot the location of the R f values and BP fragment length for each of the DNA standard fragments in well 1 Draw a “standard line” or “line of best fit” through all the plotted points

21. Repeat measurements for family DNA

22. Use your standard curve for analysis of the Smith family