1. Reverse Dot Blot for Human Mutation Detection
Dr Pupak Derakhshandeh, PhD
Ass Prof of Medical Science of Tehran University

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, untreated laboratory values are hbg <6
Thalassemia major, untreated laboratory 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
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
30 cycles
72°C for 5 min
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 color development

37. Preparation of membrane strips
Biodyne C (Pall Biomedical, U.S.A.) membrane
Membrane : activated briefly in 0·1 N HCl
Rinsed with water and soaked in 16% 1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide (EDC) for 15 min
it was rinsed in water and air dried overnight
Oligonucleotide probes were diluted with 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
Hybridized with the filter strips containing the normal and mutant probes
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
The pouch of reactants was denatured in boiling water for 5 min.
Hybridized at 428C ´ 1 h
Membrane strips were then washed 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 hybridization signal) 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 M sodium 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 times with 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 NaOH for 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 M

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
ARMS
Reverse hybridization
Turnover time
several days
6-8 hours
Equipment
Expensive (large PCR machine, gel electrophoresis, photodocumentation system)
Less expensive (small PCR machine, agarose gel, small shaking water bath)
Number of PCR reactions per sample
8-88 1
Documentation
Requires documentation process after experiment
Self-documented
Technician time (number of patients: time in days)
1:1
10:1
Starting material
Depending on the number of PCR reactions
0.5 μg genomic DNA for just one PCR reaction
Toxic materials
Ethidium bromide (carcinogen)
None

49. Reference Iranian J Publ Health, Vol. 32, No. 1, pp.11-14 (2003)
Detection of Rare and Unknown Mutations in ß- tathalassemia Traits in Iran
M Habibi Roudknar, H Najmabadi, P Derakhshandeh-Peykar, DD Farhud
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) 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 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*
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:
Lorey FW, Arnopp J, Cunningham GC. Distribution of hemoglobinopathy variants by ethnicity in multiethnic
states. Genet Epidemiol. 1996; 13:
Vetter B, Schwarz C, Kohne E, Kulozik AE. Beta- thalassemia in the immigrant and non-immigrant German populations. Br J Haematol. 1997; 97:

51. Habibzadeh F, Yadollahie M, Merat A, Haghshenas M
Habibzadeh F, Yadollahie M, Merat A, Haghshenas M. Thalassemia in Iran: an overview. Arch Irn Med. 1998; 1:
Mahboudi F, Zeinali S, Merat A, et al. The molecular basis of ß-thalassemia mutations in Fars province, Iran. Irn J Med Sci. 1996; 21: 104.
Najmabadi H, Karimi-Nejad R, Sahebjan S, et al. The ß-thalassemia mutation spectrum in Iranian population. Hemoglobin [Accepted for publication].
Newton CR, Graham A, Hepatinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acid Res. 1989; 17:
Old JM, Varawalla NY, Weatherall DJ. Rapid detection and prenatal diagnosis of beta-thalassemia: studies in Indian and Cypriot populations in UK. Lancet. 1990; 336:
Vienna Laboratories. ß-Globin Strip Assay

52. Kaufhold A, Podbielski A, Baumgarten G, Blokpoel M, Top J, Schouls L
Kaufhold A, Podbielski A, Baumgarten G, Blokpoel M, Top J, Schouls L. Rapid typing of group-a
streptococci by the use of dna amplification and nonradioactive allele-specific oligonucleotide probes. FEMS
Microbiology Letters 119: (1994) [reverse line blot hybridization]
Rapid Hla-Dpb Typing Using Enzymatically Amplified Dna And Nonradioactive Sequence-Specific
Oligonucleotide Probes Bugawan Tl, Begovich Ab, Erlich Ha
Immunogenetics 32 (4): Oct 1990
Dattagupta N, Rae PMM, Huguenel ED, Carlson E, Lyga A, Shapiro JA, Albarella JP. Rapid identification of
microorganisms by nucleic-acid hybridization after labeling the test sample. Analytical Biochemistry 177: 85-

53. Saiki RK, Walsh PS, Levenson CH, Erlich HA
Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic-analysis of amplified dna with immobilized sequence-
specific oligonucleotide probes. Proc Nat Acad Sci USA 86: (1989).
Kamerbeek J, Schouls L, Kolk A, vanAgterveld M, vanSoolingen D, Kuijper S, Bunschoten A, Molhuizen H,
Shaw R, Goyal M, vanEmbden J. Simultaneous detection and strain differentiation of Mycobacterium
tuberculosis for diagnosis and epidemiology. JOURNAL OF CLINICAL MICROBIOLOGY 35 (4):
APR 1997
Aranaz A, Liebana E, Mateos A, Dominguez L, Vidal D, Domingo M, Gonzolez O, Rodriguez Ferri EF,

54. Bunschoten AE, Van Embden JDA, Cousins D
Bunschoten AE, Van Embden JDA, Cousins D. Spacer oligonucleotide typing of Mycobacterium bovis
strains from cattle and other animals: A tool for studying epidemiology of tuberculosis. JOURNAL OF
CLINICAL MICROBIOLOGY 34 (11): NOV 1996
(According to our literature search Dattagupta et al. and Saiki et al. were the first to apply reverse dot blot,
Kaufhold et al. introduced reversed line blotting).