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Dr. Derakhshandeh, PhD

Mutation Screening. Dr. Derakhshandeh, PhD. TYPE OF MUTATIONS. WHICH TECHNIQUES DETECT WHAT TYPE OF MUTATIONS In classical genetics, three types of mutations are distinguished:. Different types of mutations. genome mutations: changes in chromosome number

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Dr. Derakhshandeh, PhD

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  1. Mutation Screening Dr. Derakhshandeh, PhD

  2. TYPE OF MUTATIONS WHICH TECHNIQUES DETECT WHAT TYPE OF MUTATIONS In classical genetics, three types of mutations are distinguished:

  3. 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

  4. genome mutations: changes in chromosome number

  5. Techniques Karyotyping, conventional cytogenetics

  6. Down Syndrome (Trisomy 21( Trisomy 2(

  7. chromosome mutations: changes in chromosome structure

  8. CHANGES IN CHROMOSOME STRUCTURE • Translocations • Large Deletions/Insertions • Inversions • Duplications/Amplifications

  9. Techniques • Conventional cytogenetics • molecular cytogenetics FISH • Molecular: • PFGE, Southern blotting, Northern BlottingFluorescence Dosage analysis • large deletions • Insertions • duplications

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

  11. gene or point mutations: mutations where changes are at molecular level

  12. Methods for detection of known mutations • Methods for detection of unknown mutations

  13. 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

  14. ARMS Amplification Refractory Mutation System Allele Specific PCR (ASPCR) PCR Amplification of Specific Alleles (PASA)

  15. 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

  16. Modifications/Adaptions to the original ARMS methodology • Multiplex ARMS

  17. 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

  18. Agarose gels showing the feasibility of the ARMS concept

  19. ARMS-PCR 1 23 45 67 89 1011 12 N M N M N M N M N M N M

  20. Methods for detection of unknown mutations

  21. Methods for unknown mutations (diagnostic methods) • These methods are relatively simple, but still require: • experience and skill to perform.

  22. BRCA

  23. BRCA1 Gene

  24. BRCA2 Gene

  25. SSCP single strand conformation polymorphism • simplicity • clearly by heteroduplex analysis (HA)

  26. Pedigree of a selected family with breast cancer

  27. SSCP AnalysisBRCA1 Exon 15, 4650delCA

  28. Pedigree of a selected family with breast cancer

  29. SSCP AnalysisBRCA1, Exon 20,Nt 5382

  30. SSCP AnalysisExon 11pi BRCA1 MS R1347G

  31. A woman having amniocentesis

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