Reverse Dot Blot for Human Mutation

Detection

Dr Pupak Derakhshandeh, PhD

Ass Prof of Medical Science of Tehran University

Introduction

Reverse dot blot (RDB) or reverse allele specific

oligonucleotide (Reverse ASO) hybridization important method for genotyping

common human mutations

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

Location of mutations in the -globin gene

Oligonucleotides used for reverse dot blot (RDB)

RDB

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

-thalassemia Patients

Molecular genetic analyses of -thalassemia

Hereditary hemoglobinopathies heterogeneous autosomal recessive

disorders -thalassemia: the most prevalent

single-gene disorder > 200 mutations in the -globin gene

located at 11p15.5 characterized by hypochromic micro

cyclic hemolytic anemia

Blood parameters of the patients and their family members

EVALUATION OF BONE INVOLVEMENT IN BETA THALASSEMIA MAJOR

Beta thalassemia minor A few oval, elliptocytes and basophilic stippling

Image 1C - Beta thalassemia minor (400 X Magnification)

                                                                                                                        

Thalassemia major, untreatedlaboratory values are hbg <6.7 hgb,20 hct, 62 MCV

Thalassemia major, untreated (250 X Magnification)

                                                                                                                        

Pedigree of the b-Thalassemia family

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

a woman having amniocentesis

Untreated Patient

affected individuals manifest failure to thrive

Shortened life expectancy

Screening for causal mutations

genomic DNA from patient blood samples

reverse dot blot (RDB) amplification refractory mutation

system-polymerase chain reaction (ARMSPCR)

DNA sequencing

PCR from genomic DNA

720 bp

Strips

N M

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10

The Blots

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

Amplicons Amplification products denatured hybridized

with mutation specific DNA probes covalently bound to solid membran

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

Materials and Methods

Total genomic DNA extracted from peripheral blood

leukocytes Amniotic fluid cells (AF) chorionic villi (CVS)

Oligonucleotide probes

A C6-amino-link phosphoramidite amino moiety on the 5' end of the

product

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

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

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

MATERIALS AND METHODS

PCR from 150 ng of genomic DNA

Preparation of membrane strips

Allele-specific hybridization and color development

Preparation of membrane strips

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.

Allele-specific hybridization and colour development

50–60 l 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)

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

Allele-specific hybridization and color development

The strips were then reacted at room temperature for15 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

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

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.

Automated DNA sequencing

Cd 2C>G

ARMS-PCR

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

PCR-RFLP1 2 3 M 4 5 6 7

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

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

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).

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