Download Lecture 7

11/18/2013
Sequencing candidate genes near linked genetic markers is used to identify mutations that cause the trait of interest
Sequences are compared against one or more reference sequences
Insertion (add one or more nucleotides)
frameshift
lost activity
Protein Activity
+
‐
A
deletion
‐
A
UUAUGUCUACUUUACGUUCCUCUCGUGGGGCAUAGUAC
MetSerThrLeuArgSerSerArgGlyAla*
+
missense (nonsynonymous) mutation
UUAUGUCUACUUUAGUUCCUCAUCGUCGGGCAUAGUAC
MetSerThrLeuValProHisArgArgAla*
UUAUGUCUACUUAAGUUCCUCAUCGUGGGGCAUAGUAC
MetSerThr*
insertion
insertion + deletion
+
‐
+
nonsense mutation
Frameshift mutations
UUAUGUCUACUUUGUUCCUCAUCGUGGGGCAUAGUAC
MetSerThrLeuPheLeuIleValGlyHisSer…
UUAUGUCUACUUUAGUUACUCAUCGUGGGGCAUAGUAC
MetSerThrLeuValThrHisArgGlyAla*
UUAUGUCUACUUUAGUUCCCCAUCGUGGGGCAUAGUAC
MetSerThrLeuValProHisArgGlyAla*
The consequences of each mutation depend on their effect and the inheritance pattern
UUAUGUCUACUUUACGUUCCUCAUCGUGGGGCAUAGUAC
MetSerThrLeuArgSerSerSerTrpGlyIleVal…
UUAUGUCUACUUUAGUUCCUCAUCGUGGGGCAUAGUAC
MetSerThrLeuValProHisArgGlyAla*
silent (synonymous) mutation
Deletion (missing one or more nucleotides)
frameshift
lost activity
UUAUGUCUACUUUAGUUCCUCAUCGUGGGGCAUAGUAC
MetSerThrLeuValProHisArgGlyAla*
Protein Activity
missense (nonsynonymous) mutation
Point mutation (single nucleotide change)
silent mutation normal protein activity
missense mutation
reduced, altered or lost activity
nonsense mutation lost activity
wt
Point mutations
wt
+
‐
Summary: Mutations at the Molecular Level I
A. Forward mutations:
1. Single nucleotide substitutions:
At DNA level: transition: purine to purine or pyrimidine to pyrimidine
transversion: purine to pyrimidine or vice versa
At protein level:
silent (synonymous) mutation: same amino acid
missense (nonsynonymous) mutation: different amino acid may or may not be functional
nonsense mutation: stop codon
2. Insertions or deletions
At protein level:
frameshift leading to:
altered protein sequence from the mutation site
premature stop codon
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Summary: Mutations at the Molecular Level II
B. Reverse mutations:
Summary: Mutations at the Molecular Level III
C. Intragenic suppressor mutations:
1. Exact reversion: AAA (lys) mutated to GAA (glu)
reversion to AAA (lys)
1. Frameshift of opposite sign at a second site
within the gene (+ to ‐ or ‐ to +) to correct the frame
2. Equivalent reversion: UCC (ser) mutated to UGC (cys)
reversion to AGC (ser)
2. Second site missense mutation: a second site
change that restores a protein with WT function
D. Extragenic suppressor mutations:
1. Nonsense suppressor
2. Missense suppressor 3. Frameshift suppressor
Diagnostic testing I: The mutation that causes the disease can be tested to determine clinical status in the prenatal or postnatal setting
tRNA mutations
Testing by Southern blot
Sickle‐cell anemia ‐‐‐ affects β‐globin chain of hemoglobin
Two alleles: HbA and HbS
HbA / HbA (Normal): red blood cell never sickles
HbA / HbS (Sickle cell trait): No anemia; red blood cells sickle only under low oxygen conditions.
HbS / HbS (Sickle cell anemia): sickle‐shaped red blood cells
A transversion point mutation causes sickle‐cell anemia
amino acid 6
Glu
CCTGAGG
Val
CCTGTGG
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Diagnostic testing 2: A genetic marker in or near the disease‐associated gene (i.e. not the actual mutation can also be used for disease diagnosis.
Problem: An unaffected couple comes in for genetic counseling. Their first child is affected with cystic fibrosis (autosomal recessive). Is their fetus predicted to be affected?
DNA Fingerprinting
With the exception of identical twins or other clones, each organism has a unique DNA sequence. Genetic markers can be used to generate a DNA pattern or “fingerprint” that can be used to identify specific individuals.
You test a genetic marker that is 1kb upstream (5’) of CFTR
Dad
Mom
+/cf
Affected
child
+/cf
7 kb
cf/cf
Fetus
? ? +
cf
5 kb
+
3 kb
cf
+
cf
What if the RF
between the
marker and the mutation is 10%?
With enough independent
markers, a pattern unique
to each individual on Earth
can be identified!
+
0.1 X 0.1 = 0.01
cf
DNA Fingerprinting
Comparing the genotypes of individuals at many different loci can determine
the identity between different pairs of samples
e.g. a crime scene sample and a group of suspects
the relationship between different individuals
e.g. reuniting family members
e.g. geneology
e.g. which man is the father?
Minisatellite (VNTR) or microsatellite (short tandem repeat) markers are used)
e.g. ATGCGGTCATCATCATCATCATTTGACCT = (CAT)n
A simple DNA fingerprinting problem
Your cat, which you had been meaning to “fix”, is now
pregnant. After she delivers her litter of kittens, you
decide to determine which of the neighborhood Toms
is responsible. You genotype a microsatellite marker
and the results are as follows:
5’-……GTACTACTACCGCACACACACACACACACACACACCCAGCTTTGAGCAC……-3’
3’-……CATGATGATGGCGTGTGTGTGTGTGTGTGTGTGTGGGTCGAAACTCGTG……-5’
Which male is the father?
How could you make the
result more robust?
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CSI
CSI
100 students attended the last LS4 lecture. A sandwich wrapper was left behind and a swab was used to recover DNA. Who left trash in LaKretz?
Clearly we need a second marker.
2A/2A 2B/2B 2A/2B wrapper
Genotype all of the students who were there
2A and 2B alleles are equally frequent in the population.
1A/1A 1B/1B 1A/1B wrapper
Alleles 1A and 1B are equally frequent in the population
p2 (1A/1A) = .5 X .5 = .25
q2 (1B/1B) = .5 X .5 = .25
2pq (1A/1B) = 2 X .5 X .5 = .5 allele 1A
allele 1B
75 of you can go home
25 suspects left
25
25
50
p2 (2A/2A) = .5 X .5 = .25
q2 (2B/2B) = .5 X .5 = .25
2pq (2A/2B) = 2 X .5 X .5 = .5
allele 2A
allele 2B
19 can go home
6 suspects left
6
6
13
f(1B/1B) X f(2A/2A)
.25 X .25 = .0625 or 1 in 16 ≈ 6 people
CSI
A third marker should do it
3A/3A 3B/3B 3A/3B wrapper
3A allele frequency is 9%
3B allele frequency is 91%
p2 (3A/3A) = .09 X .09 = .008
q2 (3B/3B) = .91 X .91 = .83
2pq (3A/3B) = 2 X .91 X .09 = .16
allele 3A
allele 3B
Gotcha! 0
5
1
f(1B/1B) X f(2A/2A) X f(3A/3B)
.25 X .25 X .16 = .01 or 1 in 100
But………..what if it was someone from the other class who attended this class?!?!?!?!?!?
Need more and better markers to zero in on a single person on the planet
Apartment complexes are genotyping each pet so they can tell if a dog owner fails to clean up after their dog!
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