1. Dr. Derakhshandeh, PhD Mutation Screening

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

3. 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. 4 genome mutations: changes in chromosome number changes in chromosome number

5. 5 Techniques Karyotyping, conventional cytogenetics

6. 6 Down Syndrome (Trisomy 21( Trisomy 2(

7. 7 chromosome mutations: changes in chromosome structure

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

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

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

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

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

13. 13 Methods for detection of unknown mutations

14. 14 DGGE Denaturing gradient gel electrophoresis  is often used in diagnostic laboratories  non-radioactive tracers and detects almost all mutations

15. 15 detection of unknown mutations  Small Mutations  Physical methods:  DGGE: eg. DMD, Thal  Single stranded conformation polymorphism analysis (SSCP)  Heteroduplex analysis (HA)

16. 16 Methods for unknown mutations (diagnostic methods)  These methods are relatively simple, but still require:  experience and skill to perform.

17. 17 PTT For BRCA1/2 using the Protein Truncation Test (PTT) for exon 11 of BRCA1 & exon 10-11 of BRCA2 These exons cover approximately over 60% of each gene

18. 18 Protein truncation test PTT

19. 19 PTT  Coding sequence without introns  cDNA via RT-PCR from RNA  or large exons in genomic DNA

20. 20 cDNA  It is PCR amplified  The forward primer carries at its 5' end a T7 promoter  followed by a eukaryotic translation initiation sequence  which includes an ATG start codon  Next is a gene-specific sequence designed so that the sequence amplified reads in-frame from the ATG

21. 21 Protein truncation test (PTT)

22. 22 PTT  After amplification  the PCR product is added to a coupled in vitro transcription-translation system  For detection a labelled amino acid is included  which is usually methionine, leucine or cysteine  The label can either be a radionucleotide such as [35S]  which is visualised by autoradiography  Or biotin which is detected by a colorimetric Western blot employing a streptavidin-biotin-alkaline phosphatase complex

23. 23 PTT  The polypeptides produced are separated by size using an SDS-PAGE gel.  If the product is only full length  no truncating mutation is present  Truncating mutations result in shorter products  the size of which gives the approximate position of the mutation.

24. 24 Protein truncation test  used in diagnostic laboratories dealing with cancer genes because they often contain truncating mutations.

25. 25 Protein truncation test (PTT)

26. 26 ADVANTAGES Detects truncating mutations:  Allows the analysis of large stretches of coding sequence (up to 5 kb: 2kb:genomic DNA, 1.3-1.6kb cDNA is best)  Either: large single exons (DNA template) or multiple exons (RNA template)  Length of the truncated protein pinpoints the position of the mutation, thereby facilitating its confirmation by sequencing analysis  SENSITIVITY: the sensitivity of PTT is good

27. 27 DISADVANTAGES  Not applicable to all genes  E.g. APC, BRCA1, BRCA2 and Dystrophin all have approximately 90-95% truncating mutations  but NF1 has only 50% truncating mutations respectively  Most powerful as a technique when RNA is used, however, most laboratories only have DNA stored

28. 28 DISADVANTAGES  The most readily available source of RNA is blood  However expression of the target gene in this tissue may below, requiring technically more demanding nested amplification reactions to obtain sufficient signal  Cannot detect mutations occurring outside the coding region, which affect control of expression and RNA stability

29. 29

30. 30 Mutation Databases

31. 31 Current mutation detection methods

32. Reverse Dot Blot for Human Mutation Detection

33. 33 Introduction Reverse dot blot (RDB) or reverse allele specific oligonucleotide (Reverse ASO) hybridization important method for genotyping common human mutations

34. 34 Commonly used in: a high mutation spectrum high frequency disorders such as: –cystic fibrosis –hemoglobin C (HbC) –hemoglobin E (HbE) –hemoglobin S (HbS) –ß-thalassemias

35. 35 Location of mutations in the  -globin gene

36. 36 Incubation nucleic acids: incubated with an enzyme conjugated to streptavidin. enzyme-conjugated, streptavidin-biotin-nucleic acid complex is then washed incubated with –a chromogenic –or luminogenic substrate, which allows visualization of hybridized spots

37. 37 Materials and Methods Total genomic DNA extracted from peripheral blood leukocytes Amniotic fluid cells (AF) chorionic villi (CVS)

38. 38 A woman having amniocentesis

39. 39 Oligonucleotide probes A C6-amino-link phosphoramidite amino moiety on the 5' end of the product

40. 40 Oligonucleotides used for reverse dot blot (RDB)

41. 41 RDB

42. 42 Reverse dot (RDB) blot hybridization for detection of 10 common β-thalassaemia mutations

43. 43  -thalassemia Patients

44. 44 PCR from genomic DNA 720 bp

45. 45

46. 46

47. 47 Strips N M 123456789123456789 1 2 3 4 5 6 7 8 9 10 … Pat1 Pat2 Pat3 Pat4 Pat5 Diff. Mutations

48. 48 The Blots

49. 49 Comparison of different factors determining the efficiency of ARMS and reverse hybridization in beta thalassemia diagnosis ARMSReverse hybridization Turnover timeseveral days6-8 hours EquipmentExpensive (large PCR machine, gel electrophoresis, photodocumentationsyst em Less expensive (small PCR machine, agarose gel, small shaking water bath) Number of PCR reactions per sample 8-881 DocumentationRequires documentation process after experiment Self-documented Technician time (number of patients: time in days) 1:110:1 Starting materialDepending on the number of PCR reactions 0.5 μg genomic DNA for just one PCR reaction Toxic materialsEthidium bromide (carcinogen) None