Dr. Derakhshandeh, PhD

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Mutation Screening

• Dr. Derakhshandeh, PhD

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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
  BlottingFluorescence Dosage analysis
• large deletions
• Insertions
• duplications

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Interphase FISH Examples
18 (aqua), X (green), and Y (red).
13 (green), and 21 (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 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 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 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