Balanced Translocation detected by FISH

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Balanced Translocation detected by FISH
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Red- Chrom. 5 probe
Green- Chrom. 8 probe
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2D Protein Gels
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MS-peptide size signature match to all predicted
proteins
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POSITIONAL CLONING
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X
2
3
Parent
X
Gamete
X
Child
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A1
B1
C1
D1
X
2
3
E2
Parent
B2
C2
A2
A2
B1
C1
D2
X
Gamete
B1
C1
A2
X
Child
10
A1
B1
C1
D1
X
2
3
E2
Parent
B2
C2
A2
A2
B1
C1
D2
X
NR
Gamete
R
B1
C1
A2
X
Child
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Positional Cloning by Recombination Mapping

• Follow the mutation
• 2. Follow which DNAs are
• co-inherited (linked)

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Positional Cloning by Recombination Mapping

• Follow the mutation
• To determine disease gene
• presence or absence (genotype)
• from phenotype you must
• first establish
• Dominant / recessive
• Aurosomal / sex-linked

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SINGLE GENE DEFECTS
Modes of Inheritance
To deduce who (likely) has one or two copies of
mutant gene
Affected Female
Unaffected Male
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/
D/
D/
/
AUTOSOMAL DOMINANT
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a/
a/
x/
/Y
x/
/Y
a/a
x/Y
RECESSIVE
RECESSIVE
X-LINKED
AUTOSOMAL
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Positional Cloning by Recombination Mapping
2. Follow which DNAs are co-inherited
(linked) Use DNA sequences that differ among
individuals within a family- Polymorphisms.
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X
2
3
Parent
X
Gamete
X
Child
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A1
B1
C1
X
2
3
Parent
B2
C2
A2
A2
B1
C1
X
Gamete
B1
C1
A2
X
Child
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Recombination Mapping
Measures distance between 2 sites on a chromosome
according to frequency of recombination
Distance between 2 DNA markers or Distance
between a disease gene and a DNA marker
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Male
Female
X
A1 B1
A1 B3
A2 B2
A1 B4
Meiosis
Gametes
Gametes
Offspring
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Female
A1 B1
w y

A2 B2
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A1 B1
w y
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A2 B2

2
A1 B2
w
Easy for Drosophila
y
A2 B1
4
Gametes
A1 / A1 white eyes B1 / B1 yellow body
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No fixed proportional
Conversion between
Genetic distance (cM)
and
Physical distance (kb, Mb)
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VNTR / STRP DETECTION
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RECOMBINANT
Heterozygosity for A and B
Recognize haplotype
Linkage (cis)
MEIOSIS
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INFORMATIVE MEIOSIS
Ideally- unambiguous inheritance of mutation
and markers (requires heterozygosity for each
in parent) knowledge of which alleles linked in
parent (phase)
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Meiosis
Meiosis
Meiosis
A2 B2
A1 B1
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FAMILY A
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Assign numbers to results of linkage
analysis to deal with non-ideal meioses to
sum data from many meioses in a family to sum
data from several families
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If unlinked-
?
If linked and RF
1/2
?
Likelihood of R
1 -
?
Likelihood of NR
1/2
Family A has 1 recombinant and 5 Non-Recombinants
?
Likelihood, given linkage of
Or given unlinked-
5
?
. (1- )
?
?
L ( )
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L (1/2) (1/2)
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Z 3
Lod
q
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FAMILY B
A1

A2
D
NR
NR
NR
NR
NR
R
R
R
R
R
R
NR
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Family B- Disease gene may be linked to A1 or A2
Consider equally likely
50 chance Family B has 1 R and 5 NR
50 chance Family B has 5 R and 1 NR
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Phase unknown
?
0.1 0.2 0.3 0.4 0.5
Z 0.28 0.32 0.22 0..08 0
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Phase known
Phase unknown
?
0.1 0.2 0.3 0.4 0.5
Z 0.28 0.32 0.22 0..08 0
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For family A with meioses 1, 2, 3, 4 ..
Z Z1 Z2 Z3 Z4 ..
For multiple families, A, B, C, D..
Z Z(A) Z(B) Z(C) Z(D) .
Assumption same gene responsible for disease
in all families Problem locus heterogeneity
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Z 3
Lod
q
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LINKAGE DISEQUILIBRIUM
Many generations
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ZOO BLOT
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PCR test DNA segments
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SSCP
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Normal
D
F506
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Testing for specific mutations
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ARMS 3 mis-match of primer
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TaqMan
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OLA
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