Reverse Dot Blot for Human Mutation Detection

1. Reverse Dot Blot for Human Mutation Detection Dr Derakhshandeh, PhD

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

4. 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

5. Location of mutations in the b-globin gene

6. Oligonucleotides used for reverse dot blot (RDB)

7. RDB

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

9. b-thalassemia Patients

10. Molecular genetic analyses of b-thalassemia • Hereditary hemoglobinopathies • heterogeneous autosomal recessive disorders • b-thalassemia: the most prevalent single-gene disorder • > 200 mutations in the b-globin gene located at 11p15.5 • characterized by hypochromic micro cyclic hemolytic anemia

13. Blood parameters of the patients and their family members

14. EVALUATION OF BONE INVOLVEMENT IN BETA THALASSEMIA MAJOR

15. Beta thalassemia minor A few oval, elliptocytes and basophilic stippling Image 1C - Beta thalassemia minor (400 X Magnification)

16. Thalassemia major, untreatedlaboratory values are hbg <6.7 hgb,20 hct, 62 MCV Thalassemia major, untreated (250 X Magnification)

18. Pedigree of the b-Thalassemia family

19. Therapy • no viable forms of treatment • a chronic course requiring repeated blood transfusions • that usually leads to iron overload • no other effective therapy is presently available • the best course: prevention through prenatal diagnosis

20. a woman having amniocentesis

21. Untreated Patient • affected individuals manifest failure to thrive • Shortened life expectancy

22. Screening for causal mutations • genomic DNA from patient blood samples • reverse dot blot (RDB) • amplification refractory mutation system-polymerase chain reaction (ARMSPCR) • DNA sequencing

23. PCR from genomic DNA 720 bp

26. Strips 1 2 3 4 5 6 7 8 9 10 N M 1 2 3 4 5 6 7 8 9

27. The Blots

28. RDB procedure • exons (or other regions of interest) • amplified by the polymerase chain reaction (PCR) • using labeled oligonucleotide primers • 5' biotin label on PCR primers

29. Amplicons • Amplification products • denatured • hybridized • with mutation specific DNA probes • covalently bound to solid membran

30. 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

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

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

33. In vitro amplification of DNA by PCR Reaction mixture: • 5 µl template DNA • 5 µl forward primer (B-F27, 5 pmol/µl) • 5 µl reverse primer (R518, 5 pmol/µl) • 2.5 µl dNTP’s (2.5 mM of each dNTP) • 5 µl 10x PCR buffer • 1.5 µl 50 mM MgCl • 0.25 µl Taq polymerase • 23.75 µl water

34. PCR program: Our forward primer is biotinylated • 94°C for 5 min • 1 cycle • 94°C for 1 min • 50-55°C for 1 min • 72°C for 1 min • 30 cycles • 72°C for 5 min • 1 cycle • 4°C hold

35. Remarks • Repeated freeze thawing of the biotin labeled oligo or PCR products may damage the biotin label • Preferably the membrane should be stripped as soon as possible, but this can also be done a few days after the hybridization. • For chemiluminescent detection, the Solution A+B should be warmed to roomtemperature for at least 30 min

36. MATERIALS AND METHODS • PCR from 150 ng of genomic DNA • Preparation of membrane strips • Allele-specific hybridization and colordevelopment • Preparation of membrane strips

37. Preparation of membrane strips • Biodyne C (Pall Biomedical,U.S.A.) membrane • Membrane : activated briefly in 0·1 N HCl • Rinsedwith water and soaked in 16% 1-ethyl-3-[3-dimethylaminopropyl] • carbodiimide (EDC) for 15 min • it was rinsed in waterand air dried overnight • Oligonucleotide probes were dilutedwith 0·5 M NaHCO3/Na2CO3 buffer, pH 8·4(0.5 pmol/ml) for application onto the membrane.

38. Allele-specific hybridization and colour development • 50–60 ml of biotinylated-PCR product • Hybridizedwith the filter strips containing the normal and mutantprobes • in 0·8 ml hybridization buffer (2 ´ SSC,0·1% sodium dodecyl sulphate) (1 ´ SSC¼0·3 M NaCl,0·03 M sodium citrate)

39. Allele-specific hybridization and color development • sealed in a cooking pouch • Thepouch of reactants was denatured in boiling water for 5 min. • Hybridized at 428C ´ 1 h • Membrane strips were thenwashed in 0·4 ´ SSC,0·1% SDS at 428C for 10 min

40. Allele-specific hybridization and color development • The strips were then reacted at room temperature for 15 min with 20 ml streptavidin horse-radish peroxidase (Gibco BRL, as conjugate for the biotin-labelled hybridizationsignal) in 20 ml 2 ´ SSC, 0·1% SDS • washes(5 min ´ 2) in 2 ´ SSC, 0·1% SDS and (2 min ´ 2) in 0·1 Msodium citrate pH 5·0

41. Allele-specific hybridization and color development • Color development was carried out: • with 0·1% 3,30,5,50-tetramethylbenzidine dihydrochloride in • 0·1 M sodium citrate and 80 ml of 3% hydrogen peroxide for • 30 min at room temperature • The reaction was stopped : • rinsing once with 0·1 M sodium citrate and several timeswith water

42. Preparation of membrane strips • Approximately 4 ml was applied to each spot • allowed to dry for 15 min before fixation in 0·5 N NaOHfor 1 min • The membrane was then rinsed thoroughly with • water and air dried overnight • Membrane strips: stored at room temperature in adesiccator for up to 6 months.

43. Automated DNA sequencing Cd 2C>G

44. ARMS-PCR

45. Haplotype analysis of the β-globin gene cluster from the patient's family.

46. PCR-RFLP 1 2 3 M 4 5 6 7

47. Direct genomic sequencing of the β-globin gene (ATG→AGG substitution of initiation codon)(a) The sequence of sense stranded sequence using Ex1 forward (b) The sequence of antisense stranded sequence using 3' reverse

48. Comparison of different factors determining the efficiency of ARMS and reverse hybridization in beta thalassemia diagnosis

49. Reference • J Clin Microbiol. 2001 March; 39(3): 871–878. Reverse Dot Blot Assay (Insertion Site Typing) for Precise Detection of Sites of IS6110 Insertion in the Mycobacterium tuberculosis Genome Lauren M. Steinlein and Jack T. Crawford* • Ian J Pub Heal. Spectrum of b-thalassemia Mutations in Isfahan Province of Iran (2007, in press) P Derakhshandeh-Peykar, H Hourfar, M Heidari, M Kheirollahi, M Miryounesi, and DD Farhud • Haemoglobin (2007, in press.) Distribution of ß-thalassemia mutations in Northern provinces of Iran. Derakhshandeh-Peykar P, Akhavan-Niaki H, Tamaddoni A, Ghawidel-Parsa S, Holakouie Naieni K, Rahmani M, Babrzadeh F, Dilmaghani-Zadeh M, Farhud DD (2007).

50. References • Lee GR, Forester J, Lukens J, Paraskovas F, Greer JP, Rodgers GM. The Wintrobe’s Clinic Hematology. Vol 1. 10th ed.Baltimore: Lippincott, Williams and Wilkins;1999. • Huisman THJ, Carver MFH. The beta- and delta-thalassemia repository. Hemoglobin. 1998; 22: 169-95. • Lorey FW, Arnopp J, Cunningham GC. Distribution of hemoglobinopathy variants by ethnicity in multiethnic • states. Genet Epidemiol. 1996; 13: 501-25. • Vetter B, Schwarz C, Kohne E, Kulozik AE. Beta- thalassemia in the immigrant and non-immigrant German populations. Br J Haematol. 1997; 97: 266-72.