Transcript Reverse Dot Blot for Human Mutation Detection

Reverse Dot Blot for
Human Mutation
Detection
Dr Derakhshandeh, PhD
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 b-globin gene
Oligonucleotides used for reverse dot blot (RDB)
RDB
Reverse dot (RDB) blot hybridization for detection
of 10 common β-thalassaemia mutations
b-thalassemia Patients
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
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, untreated
laboratory 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
1 2
NM
1
2
3
4
5
6
7
8
9
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, streptavidinbiotin-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 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)
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 for
15 min with 20 ml streptavidin horse-radish
peroxidase
(Gibco BRL, as conjugate for the biotinlabelled 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-RFLP
1
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
8-88
1
Requires documentation
process after experiment
Self-documented
per sample
Documentation
Technician time (number of 1:1
patients: time in days)
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



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).
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.
 Habibzadeh F, Yadollahie M, Merat A, Haghshenas M. Thalassemia
in Iran: an overview. Arch Irn Med. 1998; 1: 27-34.
 Mahboudi F, Zeinali S, Merat A, et al. The molecular basis of ßthalassemia mutations in Fars province, Iran. Irn J Med Sci. 1996;
21: 104.
 Najmabadi H, Karimi-Nejad R, Sahebjan S, et al. The ßthalassemia mutation spectrum in Iranian population. Hemoglobin.
2000. [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: 2503-16.
 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: 834-7.
 Vienna Laboratories. ß-Globin Strip Assay. 1998.
 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: 19-25 (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): 231-241 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 89 1989
 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: 62306234 (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): 907-914
 APR 1997
 Aranaz A, Liebana E, Mateos A, Dominguez L, Vidal D, Domingo
M, Gonzolez O, Rodriguez Ferri EF,
 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): 2734-2740 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).