dr. derakhshandeh, phd mutation screening. 2 type of mutations which techniques detect what type of...
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Dr. Derakhshandeh, PhD
Mutation ScreeningMutation Screening
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TYPE OF MUTATIONSTYPE 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 Different types of mutationsmutations
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 genome mutations:mutations:
changes in changes in chromosome chromosome
numbernumber
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TechniquesTechniques
Karyotyping, conventional cytogenetics
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Down Syndrome Down Syndrome ((Trisomy 21(Trisomy 21(
Trisomy 2(Trisomy 2(
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chromosome mutations: chromosome mutations: changes in chromosome changes in chromosome
structurestructure
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CHANGES IN CHROMOSOME CHANGES IN CHROMOSOME STRUCTURESTRUCTURE
TranslocationsLarge Deletions/InsertionsInversionsDuplications/Amplifications
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TechniquesTechniques
Conventional cytogenetics molecular cytogenetics
FISHMolecular:
PFGE, Southern blotting, Northern BlottingFluorescence Dosage analysis
large deletionsInsertionsduplications
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Interphase FISH Examples
13 (green),13 (green), and 21 (red) and 21 (red)18 (aqua),18 (aqua), X (green),X (green), and Y (red). and Y (red).
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gene or point gene or point mutations:mutations:
mutations where mutations where changes are at changes are at molecular levelmolecular level
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Methods for Methods for detection of known detection of known
mutationsmutations
Methods for Methods for detection of unknown detection of unknown
mutationsmutations
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Methods for detection of Methods for detection of known mutationsknown 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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ARMSARMS
Amplification Refractory Amplification Refractory Mutation System Mutation System
Allele Specific PCR (ASPCR) PCR Amplification of Specific Alleles (PASA)
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UsesPopulation 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 Agarose gels showing the feasibility of the ARMS feasibility of the ARMS conceptconcept
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ARMS-PCRARMS-PCR
1 2 3 4 5 6 7 8 9 10 11 12 N M N M N M N M N M N M
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Methods for detection Methods for detection of unknown of unknown mutationsmutations
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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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BRCABRCA
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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 AnalysisBRCA1 Exon 15, 4650delCA
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Pedigree of a selected family with breast cancer
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SSCP AnalysisBRCA1, Exon 20,Nt 5382
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SSCP AnalysisExon 11pi BRCA1 MS R1347G
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A woman having amniocentesis