Reverse Dot-Blot Detection of the African-American

Reverse Dot-Blot Detection of the African-American
P-Thalassemia Mutations
By P. Sutcharitchan, R. Saiki, T.H.J. Huisrnan, A. Kutlar, V. McKie, H. Erlich, and S.H. Ernbury
DNA-based diagnosis of the P thalassemias provides accuracy to newborn screening, genetic counseling, and prenatal
diagnosis. However, the use of polymerase chain reaction
(PCRI-based methods is challenged by the great number of
different P-thalassemia mutations thatexist even within defined ethnic groups. In this regard, the reverse dot-blot
method offers a means of screening for several mutations
with a single hybridization reaction. We have applied the
reverse dot-blot method to the detection of the P-thalassemia mutations of African-Americans. We used two biotinlabeled primer pairs in a duplex reaction to amplify and label
two P-globin targetDNA fragments that encompass all
known African-American P-thalassemia mutations. The PCR
products were denatured and hybridized to polyT-tailed,
membrane-fixed, allele-specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 @-thalassemiamutations and their
corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic
strip, and seven lesscommon P-thalassemia mutations were
included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these
alleles. Reverse dot-blot diagnosis of the less common Pthalassemia mutations precludes the need for alternative,
more technically challenging methods. This method provides a rapid, accurate method fordiagnosisof P thalassemia among African-Americans and other ethnic groups in
which p thalassemia occurs.
0 1995 by The American Society of Hematology.
HE P THALASSEMIAS AND@-globin hemoglobinopathies constitute majorhealth problems forpopulations
in which these disorders are prevalent."* The impact of p
thalassemia, hemoglobin(Hb) S, and Hb Calleles in the
African-American populationrelates to their frequencies and
the clinical severity of resultant syndromes. Gene frequencies in this ethnic group are 0.004 for P thalassemia, 0.045
for Hb S, and 0.015 for Hb C.' Homozygosity for P thalassemia and its coinheritance with a sickle cell gene result in
symptomatic
clinical
' One
theof
determinants of clinical severity is the extent of impairment of Hb
A production; ,F-thalassemia genes donot direct the production of Hb A and often have greater clinicalimpact than
P'-thalassemia genes, whichdirectproductionofreduced
amounts."~l' Compound inheritanceof Hb S and Hb C genes
results in Hb SC disease, avariant of sickle cell disease
having a distinct clinical nature.15.'h.'x.'"
Proper management of these disorders involves newborn
screening, genetic counseling, and prenatal diagnosis; each
of which depends on accurate diagnosis.8 Neonatal andprenatal diagnosis by traditional electrophoretic methods may
be confounded by highlevelsof
Hbanobstacleobviated by direct detection of mutant genes using DNA-based
methods.'" Polymerase chain reaction (PCR)-based methods
convey to DNA diagnosis the further advantages of convenience, sensitivity, and rapidity."~21~22 However,
most PCRbased methods addresssingle, known mutations," which is a
160 different
particular disadvantage for diagnosing the over
mutationsthat cause /3 thalassemia.27Although P-thalassemia genes are distributed selectively anlong different ethnic groups, mostpopulations have several of these mutations.7.?3 Reverse dot blotting is a PCR-based method with
T
Frvm the Department vf Medicine, Chulalongkorn Hospitul, ChulalongkornUniversity,Bangkok,Thailand; the C.P. Li Biomedical
Reseurch Corporation, Arlington, VA; the Hematology Division oj
rhe Department of Medicine, Universiry of California-Sun Fruncisco
and The San Francisco General Hospital, San Francisco, and The
Northern California Comprehensive Sickle Cell Center, San Francisco,CA; the Department of Human Genetics, Roche Molecular
Systems, Alameda, CA; and the Departments (fBiochemist~yand
Molecular Biology, Medicine, and Pediatrics,
und The Sickle Cell
Center, Medical College of Georgia, Augusta, GA.
Submitted Januay 6, 199.5; accepted April 5, 1995.
Supported in part by grants fromthe National Institutes of Health
(HL-2098.5 toS.E. and HLB-05168 t(J T.H.J.H.) andthe Chinu Medical Board of the Fuculty of Medicine. Chulalongkorn Universit.y (to
PS.).
Portions of the workwerepresentedpreviously
in ahstrucr
f o r d " and referred to in a book chapter.'
Addressreprintrequeststo
Stephen H . Embuty, MI), Building
100, Room 263, San FranciscoGeneralHospital,
San Francisco,
CA 94110.
The publication costsof this article were defrayedin part by page
chargepayment. This article must thereforebeherebymarked
"advertisement" in accordance with 18 U.S.C. seclion 1734 solely lo
indicute this fact.
0 199.5 by The American Society of Hematology.
0006-4971/95/8604-0042$3.00/0
1580
the capacity to screen several known mutations in a single
hybridization
However,
mutanumber
of
the
tions that can be included on one hybridization strip is finite," and alternative means for detecting new and unknown
mutations must be available in molecular diagnostic laboratories.
In this report, we describe the development of reverse dot
blotscontainingprobes
for the 7 most common AfricanAmerican 0-thalassemia and for the Hb S and Hb C mutations. Moreover, we havedeveloped
second
a
reverse
dot-blot strip for the detection of 7 less common AfricanAmerican P-thalassemia genes. These twostrips canbe
hybridized separately or simultaneously with duplex PCRamplified, labeled target DNA to screen for all 14 AfricanAmerican P-thalassemia mutations. This diagnosticapproach has utility for routine clinical, neonatal, and prenatal
diagnosis in the African-American population.
MATERIALS AND METHODS
African-American ~-thala.ssemiamutations. Segregatingamong
African-Americans are p"- and /?'-thalassemia gene^'^^*'.^^; 0' thalassemia genes are further subdivided into those that, when coinheror severe
ited with thesicklecellgene,producemild,moderate,
reductions in Hb A production.".'" Most African-American P-thalassemia genes areof the mild P+-thalassemia variety."'." Known point
mutations responsible for P thalassemia among African-Americans
are listed in Table
and their resultant thalassemic phenotypes7."'.'1.'~~z7
I . The mutations at -88 and -29 decrease the efficiency of tranBlood, Vol 86, NO 4 (August 151, 1995: pp 1580.1585
REVERSE DOT-BLOTOF
1581
AFRICAN-AMERICAN P THAL
Table 1. The African-American p-ThalssPemia Mutations
No.
Position
Mutation
Type
1
2
-88
-29
C-T
P’
A+G
3
Codon 6
Codon 24
Codon 30
-A
T+A
P‘
8”
P’
?P+
4
5
6
7
8
9
10
11
12
13
14
IVS 1-2
IVS 1-5
G+C
T-C
p”
G+T
P‘
Codon 61
A-T
IVS 11-1
IVS 11-848
IVS 11-849
IVS 11-849
Codon 106/107
G-A
p”
p”
C-A
A-G
A+C
+G
p”
p”
Poly A signal
T-C
P’
P’
p”
Published positions and mutations responsible for P thalassemia
among African-Ameri~ans.’~’~~’’~~~~”
scription; those at codons 6 and 106/107 create frameshifts and
premature termination of translation; that at codon 61 creates a nonsense mutation; those at IVS 1-2, IVS II-l, and IVS 11-849 ablate
RNA processing; and those at codons 24, IVS 1-5, IVS 11-848, and
at the poly A signal impair RNA processing? It is assumed that the
codon 30 mutation affects RNA splicing, but its mechanism and
phenotype are unknown? The mutations at -29 and -88 account
for 80% ofthe P thalassemia among African-Americans””. We
have not included in our assessment an -87 C ”+ A Yugoslavian
mutationz8 that has also been observed in one African-American
~ubject?~
Design and synthesis of probe pairs. Careful design of probe
sequence and application of optimal quantities of probes to hybridization membranes are essential to developing reverse dot-blot methodology. Thermodynamic consideration^'^^'^ were the basis for the
design of the probe pairs shown in Table 2 that were used to discriminate between variant and wild-type sequences for Hb S, Hb C,
and 14 African-American P-thalassemia mutations. Oligonucleotides
were synthesized using phosphoramidite chemistry.’*
For closely adjacent mutations, a single normal probe was used;
ie, one normal probe was used for the Hb S, Hb C, and codon 6
-A frameshift mutation; one normal probe was used for the IVS I1 and IVS 1-5 mutations; and one normal probe was used for the
two IVS 11-848 and the IVS 11-849 mutations.
Poly-T tailing of oligonucleotide probes. Oligonucleotide probes
(200 pmol) were tailed overnight at 37°C in a 100-pL reaction mixture of 100 mmol/L K cacodylate, 25 mmoVLTris-HC1, 1 mmoU
L CoC12, and 0.2 mmol/L dithiothreitol (pH 7.6) with 160 nmol
deoxythymidine triphosphate and 60 U of terminal deoxyribonucleotidy1 transferase (Ratliff Biochemicals, Los Alamos, NM). Reactions
were stopped by the addition of 100 pL of IO mmoVL EDTA. The
length of poly-T tails was monitored by agarose gel electrophoresis
to insure that tails of approximately 400 residues were obtained.
Tailed oligonucleotides were diluted into 100 pL TE (10 mmoll
L Tris-HC1, 0.1 mmol/L EDTA [pH 8.01 ) for application to a nylon
membrane (Biodyne-B; Pall, Glen Cove, NY) using a spotting manifold (The Convertible; GIBCO-BRL, Gaithersburg, MD). Damp filters were placed on TE-soaked paper within a UV light box (Stratalinker 1800; Stratagene, La Jolla, CA) and were irradiated at 254
nm, 600 mJ/cm2 to a dose controlled by the internal metering unit
of the light box. Filters were rinsed to remove unbound oligonucleotides using 5X SSPE (1 X SSPE: 180 mmoVL NaC1, 10 mmoVL
NaH2P0,, l mmoVL EDTA [pH 7.21)with 0.5% sodium dodecyl
sulfate (SDS) for 30 minutes at 55°C and were then used immediately
for hybridization or were air dried and stored at room temperature.
The quantities of probes necessary to distinguish series of mutant
and normal alleles were tested and adjusted by trial and error by
hybridizing with control DNA samples that were amplified and labeled as described below. The quantities of each applied to filters
in picomoles is shown in Table 2.
Amplifcation and labeling of rarget DNA. Initially, we amplified
the entire &globin gene as a 1780-bp fragment using a single PCR
primer pair.” However, the use of this fragment in reverse dot blotting did not allow us to distinguish between mutant and normal
sequences, apparently because of the effect of secondary structure
on hybridization. Hence, we devised two pair of PCR primers that
directed simultaneous amplification of two P-globin DNA fragments
(duplex PCR) that encompass all the known P-thalassemia mutations, as shown in Fig 1.
The leftward primer pair consisted of: upstream primer, 5‘ BiotinAACTCCTAAGCCAGTGCCAGAAGA 3’; and downstream
primer 5’ Biotin-TCA’ITCGTCTGTCCCATTCTAAAC 3‘. The
rightward primer pair consisted of upstream primer, 5’ Biotin-TATCATGCCTCTGCACCA’ITCT 3‘; and downstream primer, 5’
Biotin-CACTGACCTCCCACATTCCCm 3’. Phosphoramidite
biochemistry3*was used to synthesize these four primers, and the
5’ terminal base of each was converted during DNA deprotection
to a primary amine-containing base that was subsequently biotinylated, as described.24These four primers were used simultaneously
in the same test tube for duplex PCRinwhich the leftward pair
directed amplification of a 774-bp fragment and the rightward pair,
a 574-bp fragment, as shown in Fig 2. In the amplification reaction,
target DNA (0.5 pg) wasamplified in a 100-pL reaction mixture
[8.3 mmol/L (NH,),SO,; 33.5 mmoVL Tris HCI; 3.3 mmolL MgCI2;
2.5 mmol/L P-mercaptoethanol; 3.4 pnoVL Na2EDTA; 400 mmoll
L each deoxyadenosine triphosphate, deoxycytidine triphosphate,
deoxyguanosine triphosphate, and deoxythymidine triphosphate; 0.2
mmoln of each biotinylated primer; and 4.0 U of Taq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CT)]. Reactions wereperformed in a programmable thermal cycler (Perkin Elmer-Cetus) at
95°C for 30 seconds, at 60°C for 3 seconds, and at 72°Cfor 3 seconds
by the Step-Cycle program for 35 cycles. Samples were incubated
at 72°C for an additional 5 minutes.
Hybridization ofjilter-bound probeswith amplified, labeled DNA.
Each filter with bound oligonucleotide probes was placed in 3 mL
hybridization solution of 4X SSPE and 0.5% SDS. Amplified DNA
in 20 pL was denatured with an equal volume of 400 mmom NaOH
with 10 mmoVL EDTA for 1 to 5 minutes, added immediately to
the 3 mL hybridization solution, and incubated at 55°C for 30 minutes. The stringency and temperature of the reaction are critical to
the success of this method. The filters were briefly rinsed once in
1X SSPE and 0.2% SDS at room temperature and once in 2 x SSPE
and 0.2% SDS at 55°C for 10 minutes. They were then incubated
in 3 mL of 1 X SSPE and 0.2% SDS solution with 0.3 pL of streptavidin-horseradish peroxidase conjugate (1 mg/mL; Vector Laboratories, Burlingame, CA) for 10 minutes at room temperature, after
which they were briefly washed 3 times in 1 X SSPE and 0.2% SDS
at room temperature. Finally, the filters were washed 3 times in 100
rnmol/L sodium citrate, pH 5.0. Color development was performed
by incubating the filters atroom temperature in thedark for 30
minutes in freshly prepared solution containing 0.1 mg/mL of
3.3’3.5’ tetramethylbenzidine, 0.003% H2O2in 100 mmol/L sodium
citrate (pH 5.0). The filters were washed twice for 5 minutes by
shaking in distilled water.
RESULTS
Results of reverse dot-blot tests are dictated by the target
DNA that hybridizes only to probes complementary to se-
1582
SUTCHARITCHAN ET AL
Table 2. The Oligonucleotide Probes and Quantity (pmol) of Each Applied t o the Filters
PositionSequence
-88
Probe
Mutation
C-T
29
A-G
Codon 6 (Hb C)
G-A
Codon 6 (Hb S )
A-T
1
6
CodonNormal
-A
1
Codon 24
T-A
Codon 30
G-C
IVS 1-2
T-C
IVS 1-5
G-T
Codon
A-T
IVS 11-1
G-A
IVS 11-848
C-A
IVS 11-849
A-G
IVS 11-849
A-C
Codon 1061107
Normal
+G
Poly A
T-C
Quantity
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
C
C
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Normal
Mutant
Mutant
Normal
Mutant
quences therein. In the normal pattern, normal DNA hyhridizes only to probes complementary tonormal sequences. For
the homozygous pattern, DNA homozygous for a particular
mutation hybridizes to the probe complementary to that mutation but not itsnormal analog and to all othernormal
probes but not their mutant analogs. In the simple heterozygous pattern, DNA with a single copy of a particular mutation hybridizes to the mutant probe and its normal analog
and to all other normal probes but not their mutant analogs.
For the compound heterozygouspattern, DNA containing
one copy each of two different mutations hybridizes to both
mutant probes and both their normal analogs and to all other
normal probes but not their mutant analogs. Amore complex
6
30 2
-29 -88
61
5' AACCCTAGGGTGTGGCTC 3'
5' CAACCCTAGGATGTGGCTC 3'
5' GGCTGGGCATAAAAGTCAGG 3'
5' TGACTTTCATGCCCAGCC 3'
5' CTCCTGAGGAGAAGTCTG 3'
5' CTCCTAAGGAGAAGTCTGC 3'
Same as Hb
5' CTCCTGTGGAGAAGTCTG 3'
Same as Hb
5' AGACTTCTCCCAGGAGTC 3'
5' AGGGCCTCACCACCAACCC 3'
5' CTCACCTCCAACTTCATC 3'
5' CTGGGCAGGTTGGTATCA 3'
5' CTGGGCACGTTGGTATCA 3'
5' CCTTGATACCAACCTGCCCAG 3'
5' GGGCAGGCTGGTATCAAG 3'
Same as IVS 1-2
5' TGGGCAGGTTGTTATCAA 3'
5' CTAAGGTGAAGGCTCATGG 3'
5' CCTAAGGTGTAGGCTCATG 3'
5' AGGGTGAGTCTATGGGAC 3'
5' GAACTTCAGGATGAGTCTATG 3'
5' ATCTTCCTCCCACAGCTC 3'
5' ATCTTCCTCCCAAAGCTC 3'
Same as IVS 11-848
5' ATCTTCCTCCCACGGCTC 3'
Same as IVS 11-848
5' ATCTTCCTCCCACCGCTC 3'
5' ACAGCTCCTGGGCAACG 3'
5' GCACGTTGCCCCAGGAG 3'
5' CATCTGGATTCTGCCTAATAAAAAA 3'
5' TCTGGATTCTGCCTAACAAAAAA 3'
848
106/107
849
849
~.
-.
1
6
IVS 2
774 bp
-+"""""""""""""""""
4-
4
4
4
4
4
4
1
4
1
4
4
4
4
4
4
4
4
4
4
4
4
4
2
1
pattern is observed with closely adjacent mutations in which
the sameoligonucleotideserves
as probeforthe
normal
sequence; with either homozygosity for oneof the mutations
or compound heterozygosity for both, there will he no hybridization to the shared normal probe.
Results from the reverse dot-blot tests for the seven most
common African-American P-thalassemia mutations and the
structural mutations Hb S and Hb C are shown in Fig 3. On
each filter strip the normal probes are located above, and the
mutant probes are below. The mutations represented by each
probe pair are listed above the panel, and the DNA samples
tested are shown on the right. The predicted hybridization
pattern of normal DNA is shown in the top strip. Homozy-
24
56
5' FLANKING
1
4
2
4
1
4
@
&
.@3'"T
<$$..&**g%
~"""""""""""-
574 bp
POLY A
'\
AAAAA
43'
-FLANKING
Fig 1. A diagram of the p-globin gene. This diagram shows the three exons (EX), t w o introns IIVSI, polyadenylation (poly A) site, and 5'
and 3' untranslated (UT) and flanking sequences of the p-globin gene. The positions of the t w o amplification primer pairsare shown as t w o
pairs of opposing arrows below the gene. Their PCR products are indicated as dotted lines, and their sizes are indicated as base pairs (bp).
The positions of the mutations responsible for p thalassemia are shown above the gene.
1583
REVERSE DOT-BLOT OF AFRICAN-AMERICAN 4 THAL
M
1
3
2
4
5
Fig 2. The /.?-globin DNA
products of duplex PCR. A UVilluminated photograph of an
ethidium bromide-permeated,
2% agarosegel on which the
774-bp products of the leftward
primer pair andthe 574-bp products of the rightward primer pair
amplified byduplex PCR were
electrophoretically
separated.
Size markers are shown in lane
M, and PCR products synthesized from 15 different DNA samples are shown lanes
in
1
through 15.
6
7
8
9
1 0 1 1 1 2 1 3 1 4 1 5
tn4bp
t !34 bp
gous hybridization patterns are observed withDNAfrom
subjects that are homozygous for the -88 C T and the
-29 A + G &thalassemia alleles. DNA homozygous for
the Hb S gene bound to the PSprobe but not to its normal
analog nor to other mutant probes but did bind to all other
normal analogs (except for Hb C which is the same as the
Hb S normal probe). The simple heterozygous pattern is
seen with the heterozygous codon 24 T + A P-thalassemia
sample. The compound heterozygous pattern was seen with
DNA containing the PSgene and a &thalassemia gene (ie,
IVS 1-5 G + T, IVS 11-849 A + G , IVS 11-849 A -t C, codon
106-107 +G).The filter strip shown in Fig 3 with its 7 0thalassemia alleles detects approximately 90% of AfricanAmerican &thalassemia mutations“’.” and the Hb S and Hb
C genes.
We developed a second reverse dot-blot strip for 7 less
common African-American &thalassemia mutations and included p5and pc (Fig 4). Again, on each filter strip the
normal probes are located above and the mutant probes are
below. The normal DNA patternis seen in the top strip.
DNA from a subject with Hb SC disease hybridized to the
p” and p’ probes, but not to their common normal analog,
and hybridized to no other mutant probe, but to all other
normal probes except for the codon 6 -Anormal probe,
which was also the probe common to Hb S and Hb C. The
pattern of hybridization for simple heterozygosity was seen
for the codon 6 -A, codon 30 G -t C, codon 61 A T, and
IVS 11-848 C + A samples. The compound heterozygous
pattern was seen with DNA containing the Hb S and IVS I2 T + C mutation, the Hb S and IVS 11-1 G A mutation,
and the Hb C and poly A T + C mutation. The data shown
in Figs 3 and 4 demonstrate that the filterswe have developed
will detect the 14 African-American P-thalassemia mutations, the Hb S and Hb C mutations, and their combinations.
+
+
+
DISCUSSION
We report a reverse dot-blot method for detecting the
known African-American &thalassemia mutations and the
Hb S and Hb C genes using either two or a single hybridization reaction. The basis of this method is to detect which of
several known mutations exists in target DNA by determin-
ing whether mutant or normal membrane-fixed probes capture PCR-amplified and labeled target DNA.” Primers were
developed for simultaneous amplification of two P-globin
gene fragments (duplex PCR) that encompass all the known
&thalassemia mutations of African-Americans. The number
of mutations screened in one hybridization is limited by the
reaction conditions required to distinguish numerous allelic
pairs. Notwithstanding the large number of mutations that
can be screened with a single reverse dot-blot rea~tion,’~.’~.”
alternative diagnostic methods such as denaturing gradient
gel ele~trophoresis“~
andDNA sequence analysis”’ should
be used for detecting the mutations not represented on the
initial reverse dot-blot. Our alternative approach was a second reverse dot-blot strip that included the 7 less common
African-American &thalassemia mutations and Hb S and
Hb C. The two strips can be hybridized simultaneously or
separately.
The ability to distinguish normal from mutant alleles and
to accurately identify the P-thalassemia mutations present
are requisites for reverse dot-blot diagnosis of 0 thalassemia.
The first challenge in developing this test is to design the
sequence and sense of both oligonucleotides3”“ and apply
the two probes to filters in amounts that allow accurate allelespecific hybridization. The quantitative ratio of the two
probes that consistently discriminates normalandmutant
alleles is determined by trial and error. The next task is to
determine, again by trial and error, the relative amounts of
probe pairs thatwill establish whichnormalandmutant
alleles are present. We have shown in Table 2 the amount
of each probe that we applied to the filtersto accurately
distinguish these P-thalassemia mutations. The accuracy and
reliability of the method is dependent on the stringency and
temperature of the hybridization step, which we optimized
at 55°C. A potential confounding influence relates to the
quantitatively unequal amplification of the two P-globin
DNA fragments (see Fig 2) that could generate signals of
different intensity from equal alleles. This vagary is redressed by the initial emprical quantifications of probe ratios
required for accurate diagnosis at each locus. A related pitfall
could result from sequence variations in target DNA that
interfere with PCR primers in directing optimal amplification
SUTCHAAITCHAN ET AL
of the two fragments. Quantitative amplification of the two
fragments is monitored by visual inspection of PCR products
in ethidium bromide gels after electrophoresis, as in Fig 2.
Accurate diagnosis is essential for genetic counseling and
prenatal diagnosis, which are mainstays of sickle cell disease
management.' Traditional electrophoretic methods for the
diagnosis of p thalassemia and hemoglobinopathies may be
complicated by complex inheritance patterns or by the presence of large amounts of Hb F. These obstacles canbe
circumvented by using DNA-based diagnostic methods..'"~."
Multi-allele screening with the reverse dot-blot method" is
6 8
n n
3 3
HbCIHbS
Codon61HbA
Codon 30 I Hb A
HbSINSI-2
Normal
Codon 61I Hb A
Homozygous-88
IVSII-1 lHbS
Homozygous -29
IVSII-8481HbA
Homozygous HbS
PolyAIHbC
Codon 24I Hb A
IVS 1-5 / Hb S
Fig 4. Reverse dot-blot strips after hybridization and color
development. These strips contain hybridization probes complementary
t o seven less common African-American /%thalassemia mutations
and the HbS and Hb C alleles. For further details see the legend t o
Fig 3.
IVS 11-849 I Hb S
A+G
IVS 11-849 / Hb S
A+C
Codon 106-107 I Hb S
Fig 3. Reverse dot-blot strips after hybridization and color
development. Hybridization probes complementary t o t h e seven most
common African-American p-thalassemia mutations and the structural mutations Hb S and Hb C are fixed t o these reverse dot-blot
strips. On each filter strip, the normalprobes are at the top and the
mutant probes are at the bottom.
The mutations represented by each
probe are listed above, and the DNA samples tested are shown on
the right. The presence of a color signal at each position indicates
the presence in amplified target DNA of sequences complementary
to thatspecific probe.
particularly useful for diagnosing p thalassemia because of
the vast diversity of P-thalassemia mutation^.^.^^ The 14 pthalassemia genotypes we have t e ~ t e d ~ . ' " .account
' ' . ~ ~ for the
several P-thalassemia phenotypes".'4.'7 present among African-Americans. The reverse dot-blot method described here
will facilitate the accurate diagnosis, genetic counseling, and
prenatal diagnosis of individuals bearing these genes. In addition, this method will provide an excellent means of confirmatory testing for newborn screening programs.
ACKNOWLEDGMENT
We acknowledge the generous support of Dr N.Mohandas (Lawrence Berkeley Laboratories), the provision of oligonucleotides by
Dr C. Levinson (Roche Molecular Systems), and the technical advice
of Mr G . Kropp (C.P. Li Biomedical Research Corp).
REVERSE DOT-BLOT OF AFRICAN-AMERICAN
THAL
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