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 by Recombination Mapping

• Follow the mutation
• 2. Follow which regions of
• DNA 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.
C
A
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VNTR / STRP DETECTION
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A1
A1
A1
A2
A3
A3
A2
A4
A4
A3
A3
A1
A4
A2
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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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No fixed proportional
Conversion between
Genetic distance (cM)
and
Physical distance (kb, Mb)
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FAMILY A
D
D
D
D


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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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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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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 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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PCR test DNA segments
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Testing for specific mutations
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ARMS 3 mis-match of primer
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OLA
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Family Trio SNP genotypes reveal haplotypes
Aa BB CC DD Ee FF Gg HH II JJ
AA BB CC Dd Ee FF GG HH II Jj
Mother
Father
AA BB CC Dd Ee FF Gg HH II JJ
Son/Daughter
A B C d E/e F G H I J
A B C D E/e F g H I J
a B C D e/E F G H I J
A B C D e/E F G H I j
Deduced haplotypes- ignoring recombination
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Creation of variant sequences Rearrangement of
sequence variants by recombination First,
consider just the creation of variant sequences
within a short stretch of DNA where there is no
significant rearrangement due to recombination
(an assumption that turns out to be valid)
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b bq bqk bqkh
g ga gar garn
f
ABCDEFGHIJKLMNOPQRST
AbCDEFGHIJKLMNOPQRST
ABCDEFgHIJKLMNOPQRST
AbCDEFGHIJKLMNOPqRST
aBCDEFgHIJKLMNOPQRST
AbCDEFGHIJkLMNOPqRST
AbCDEFGhIJkLMNOPqRST
ABCDEfGHIJKLMNOPQRST
aBCDEFgHIJKLMNOPQrST
aBCDEFgHIJKLMnOPQrST
History
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b bq bqk bqkh
g ga gar garn
f
ABCDEFGHIJKLMNOPQRST
AbCDEFGHIJKLMNOPQRST
ABCDEFgHIJKLMNOPQRST
AbCDEFGHIJKLMNOPqRST
aBCDEFgHIJKLMNOPQRST
AbCDEFGHIJkLMNOPqRST
AbCDEFGhIJkLMNOPqRST
ABCDEfGHIJKLMNOPQRST
aBCDEFgHIJKLMNOPQrST
aBCDEFgHIJKLMnOPQrST
Retention amplification of only a few haplotypes
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For any short region of DNA typically only 4-6
haplotypes are found in a sampling of present day
humans (of the many millions that must have
existed in at least one copy en route). These
local haplotypes provide some information about
ancestry. Now consider how the major haplotypes
of each short region of DNA are associated with
neighboring haplotypes to see where recombination
events took place.
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aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
High LD regions?
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aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
aBCDEFgHIJKLMnOPQrSTUVwXyZ??????????????
High LD segment
High LD segment
Recombination hot-spot
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85 of genome made up of 5-20kb high LD
blocks Only 4-5 different major haplotypes per
block in the world!
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Haplotype blocks
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100 kb
1 2 3 4 5 6 7 8
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Disease No disease /2,000 /3,000
Minor allele frequency
SNP-2a 93 130 SNP-2b 21 27 SNP-3a 140 62
SNP-3b 24 35 SNP-3c 140 260 SNP-3d 87 120
. . . . . . . . ..
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