Download STRs and Marker Analysis

DNA marker analysis
Mrs. Stewart
Medical Interventions
Central Magnet School
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 Cancer Categories
Sporadic
Hereditary
Familial
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.
BRCA 1 and BRCA 2
Tumor suppressor genes
Repair DNA damage and control cell
growth
Proto-oncogenes
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
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.
Family Pedigree
Draw a pedigree for the Smith family to represent the
mutated BRCA2 gene.
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
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.
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.)
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.
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
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
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.
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
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.
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
Table One:
Data
DNA Size
Fragment
Markers
Length in
Distance Migrated
Base Pairs
Distance
Distance to
Rf
Migrated
Reference
(mm)
- Measure
the distance in mm
Point (mm)
A÷B
well to (mm)
each
fragment
in
the
standard
A
B
from the sample
lane. Record in Table One
Fragment 1
Distance
Fragment
2
1353
to 1078
Reference Point - Measure the distance
from the sample well to the end of the gel. Record in
Fragment 3
872
Table One.
Fragment 4
603
This number
will be the same for each size marker
Fragment
5
310
fragment.
Fragment 6
281
Calculate the Rf of each fragment and record in Table
Fragment
234
One. 7
Fragment
8
194 to the nearest one hundredth.
Round
values
Plot your standard curve
Plot the location of
the Rf 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
Repeat measurements for
family DNA
Use your standard curve for
analysis of the Smith family