Mutation Screening
Dr. Derakhshandeh, PhD
TYPE OF MUTATIONS
WHICH TECHNIQUES DETECT
WHAT TYPE OF MUTATIONS
In classical genetics, three
types of mutations are
distinguished:
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Different types of mutations
 genome mutations: changes in chromosome
number
 chromosome mutations: changes in
chromosome structure
 gene or point mutations: mutations where
changes are at molecular level
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genome
mutations:
changes in
chromosome
number
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Techniques
Karyotyping, conventional cytogenetics
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Down Syndrome (Trisomy 21(
Trisomy 2(
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chromosome mutations:
changes in chromosome
structure
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CHANGES IN CHROMOSOME
STRUCTURE
Translocations
Large Deletions/Insertions
Inversions
Duplications/Amplifications
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Techniques
Conventional cytogenetics
 molecular cytogenetics
FISH
Molecular:
PFGE, Southern blotting, Northern Blotting
Fluorescence Dosage analysis
large deletions
Insertions
duplications
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Interphase FISH Examples
13 (green), and 21 (red)
18 (aqua), X (green), and Y (red).
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gene or point
mutations:
mutations where changes
are at molecular level
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Methods for detection of
known mutations
Methods for detection of
unknown mutations
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Methods for detection of known
mutations
 PCR and size separation eg. DMD
 PCR and restriction enzyme digestion eg. SMN exon 7
&8
 Allele specific amplification (ASA)
 Allele refactory mutation system (ARMS) eg. CF
 Allele specific oligonucleotide hybridisation (ASO)
 Dot Blot eg. CF
 DNA chips eg. Brca1
 Genomic DNA sequencing
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ARMS
Amplification Refractory Mutation
System
Allele Specific PCR (ASPCR)
PCR Amplification of Specific Alleles (PASA)
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Uses
Population screening
 rapid (1 working day)
 inexpensive
 non-isotopic
Used for testing for
 B-thalassaemia
 Cystic Fibrosis
 Sickle-cell anaemia
 Phenylketonuria
 Apolipoprotein E, etc
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Modifications/Adaptions to the
original ARMS methodology
Multiplex ARMS
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To set up multiplex ARMS
Determine commonest mutations in
the population
develop the muliplex ARMS these
mutations
validate the results of the multiplex
test on samples with another
methology
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Agarose gels showing the
feasibility of the ARMS concept
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ARMS-PCR
1
N
2
M
3
N
4
M
5
N
6
M
7
N
8
M
9
N
10
M
11
N
12
M
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Methods for detection of
unknown mutations
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Methods for unknown mutations
(diagnostic methods)
These methods are relatively simple,
but still require:
experience and skill to perform.
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BRCA
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BRCA1 Gene
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BRCA2 Gene
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SSCP
single strand conformation polymorphism
simplicity
clearly by heteroduplex analysis (HA)
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Pedigree of a selected family with
breast cancer
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SSCP Analysis
BRCA1 Exon 15, 4650delCA
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Pedigree of a selected family
with breast cancer
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SSCP
Analysis
BRCA1, Exon 20,
Nt 5382
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SSCP Analysis
Exon 11pi BRCA1 MS R1347G
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A woman having amniocentesis
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