Detection of Hereditary Breast
Cancer
Breast Cancer Genes
• Two genes associated with inheritance of
breast cancer
– Breast cancer gene 1 (BRCA1) – Chromosome 17
– BRCA 2 – Chromosome 13
• When normal…
– Both are tumor suppressor genes
• Repair damage and prevent cancer cells from forming
• When mutated…
– Lead to breast or ovarian cancer
BRCA 1 and BRCA 2
• 5 to 10% of all breast cancers are because of
these genes
• Women…
– 12% chance of developing breast cancer in
lifetime
• Goes up to 85% if said woman has mutated BRCA 1 or 2
• If male has BRCA 2 mutation
– 1 in 10 ratio of getting breast cancer (100,000 jump)
Family Members
• Judy is worried!!!!!
• Jennifer has DNA sequencing…
– Jennifer had breast cancer
• Test positive of BRCA 2 (negative for BRCA 1)
– Laura
• Tests positive for same mutation as Jennifer
BRCA2
• We will be looking at BRCA2
– Contains 80,000 nucleotides
• 600 mutations associated with BRCA2
– Most cause increased incidence of breast cancer (not all)
• Most of these mutations are insertion or deletion
Marker Analysis
(Haplotyping)
• Due to expense of DNA sequencing…
– We will use marker analysis to test Jennifer and Diana
• Marker analysis…
– Genetic test
– Gene mutation is analyzed using a genetic marker
• Instead of analyzing genet itself
• Genetic marker: short sequence of DNA
associated with a particular gene or trait with a
known location on a chromosome
Short Tandem Repeats
• Genetic markers used in marker analysis are short
DNA sequences
– Also called microsatellites
• STR = region of DNA composed of a short
sequence of nucleotides repeated many times.
– Number of repeated STRs varies from person to
person
• Different number of repeats = different alleles
• Most occur in introns (non-coding DNA)
– Do not affect gene function
STR for BRCA 2
• Location = chromosome 13
• STR analysis for this lab
– It is on 13, next to BRCA 2 gene
Gel Electrophoresis
• Different STRs have different repeats…
– Gel will separate alleles based on number of
repeats
• More repeats travels less
• Less repeats travels more
Loading Samples
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5 uL: 130 Volts for 30 minutes
Lane #1: DNA Size Markers
Lane #2: Helen’s DNA
Lane #3: Harold’s DNA
Lane #4: Susan’s DNA
Lane #5: Adam’s DNA
Lane #6: Negative Control
Determining Size of DNA Fragment
• Using a ruler
– Measure (ON EACH BAND!!!!)
• Distance of DNA fragment from origin (gel well)
• Distance from the origin (gel well) to the tracking dye
Calculate the Rf value
Distance the DNA fragment has migrated from the origin (gel well)
Rf =
Distance from the origin (gel well) to the reference point (tracking dye)
DNA Size Markers
Fragment Length in Base
Pairs
Fragment 1
1353
Fragment 2
1078
Fragment 3
872
Fragment 4
603
Fragment 5
310
Fragment 6
281
Fragment 7
234
Fragment 8
194
Distance Migrated (mm)
A
Distance to Reference
Point (mm)
B
Rf
A÷B
DNA Sample:
Diana
Fragment:
Fragment 1
Fragment 2
Jennifer
Fragment 1
Fragment 2
Laura
Fragment 1
Fragment 2
Judy
Fragment 1
Fragment 2
Distance Migrated (mm)
A
Distance to Reference
Point (mm)
B
Rf
A÷B
DNA Sample:
Fragment:
Diana
Fragment 1
Fragment 2
Jennifer
Fragment 1
Fragment 2
Laura
Fragment 1
Fragment 2
Judy
Fragment 1
Fragment 2
Fragment Length (in base pairs)
Allele Present:
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
Questions
1. Which allele is associated with the BRCA2
mutation? Explain your answer.
2. Which family members have the BRCA2
mutation? Explain your answer.
3. Explain whether you think Judy’s family
occurrences of breast and ovarian cancers
are sporadic, hereditary, or familial.
4. Is Judy a good candidate for BRCA1 or BRCA2
genetic testing? Explain your answer.