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2.

MATERIALS AND METHODS


2.1: Sample collection:
Whole blood samples of 100 thalassemia Intermedia patients were collected in
EDTA. Information of each patient regarding their age, sex, ethnic group, transfusion
started and clinical appearance was noted at the time of sample collection.

2.2: Inclusion criteria:


The criteria followed for the inclusion of the patients for this study was;
1. Patient was known thalassemic
2. Age at commencement of transfusion was more than three years
3. The interval between the transfusion was at least 1 month

2.3: Complete blood count:


Complete blood counts of all the samples were determined that includes the
hemoglobin estimation, red cell count, total leukocyte count, mean cell volume
(MCV), mean corpuscular hemoglobin (MCH) and mean cell hemoglobin
concentration (MCHC).These parameters were measured on automated cell
counter (Sysmex K4500).

2.4: GENE ANALYSIS :


Gene analysis for the detection of different mutations was carried out. The steps
included were
1. DNA extraction
2. PCR
3. Electrophoresis

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2.4.1: DNA extraction :


Extraction of DNA from the whole blood was carried out using the genomic DNA
purification kit by Gentra. The steps followed were as follow
a. 300 l of the whole blood was taken in 1.5 ml microfuge tube containing 900 l of
RBC lysis solution and incubated for 10 minutes at room temperature
b. Centrifuged for 20 seconds at 13,000 16,000 rpm.
c. The supernatant was removed leaving behind the visible white cell pellet.
d. The tube was vortexed vigorously to resuspend the cells.
e. 300 l of cell lysis solution was added to the resuspended cells
f. 100 l protein precipitation solution was added and vortexed vigorously till the
precipitates appeared
g. The tube was then centrifuged at 13000 16000 rpm for 3 minutes
h.The supernatant containing DNA was poured into a clean 1.5ml microfuge tube
containing 300 l of 100% Isopropenol
i. The tube was inverted gently until the thread like structure of DNA was visible
j. The tube was then centrifuged at 13000 rpm for 1minut and supernatant was removed
k. 300 l of 70 % ethanol was added and inverted several times to wash the DNA pellet
l. The tube was centrifuged at 13000 rpm for 1 minute, ethanol was discarded, the tube was
inverted and drained on a clean absorbent paper and allowed to dry in air for 10
minutes.
m. 20 l of DNA hydration solution was added to dissolve the extracted DNA
n. DNA was stored at 4oC until analysis.

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2.4.2: PCR : Amplification Refractory Mutation System


PCR method known as amplification refractory mutation system (ARMS) (Newton et.
Al. 1989) was carried out. The target DNA was amplified using the primer
complementary to the - chain mutations. The samples in which the mutations were
detected, were then tested with the primers complementary to the normal sequence of the
specific mutation to establish the homozygous and heterozygous state of the patient.
Samples giving positive reaction with the normal primer was then tested for other
mutations. Another set of primers was used to amplify a 861 bp fragment of the distal
part of the - globin gene, to serve as an internal control for PCR.

CONDITIONS FOR ARMS:


PCR for the - chain mutations was carried out using 2 l of DNA. The amplification on
the thermal cycler was carried out consisting of 25 cycles; each cycle consisted of
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denaturation at 94oC for 1 minute, primer Anneling at 65 C for 1 minute and DNA
extension at 72oC for 2 minutes. In the final cycle the extension was prolonged for
another 3 minutes. Electrophoresis of the amplified product was carried out on 6%
polyacrylamide gel. The sequence of the primers used for the detection of mutations and
the normal primers are given in table- 2.1 and 2.2 respectively.

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Table- 2.1: The sequences of the primers used for the identification of mutations
by ARMS method.
Mutation

Primer
CTC CTT AAA CCT GTC TTG TAA CCT TGT TAG

Used
with
3

Fragme
nt size
285

IVSI-5 (G-C)
Fr 8 9 (+G)

CCT TGC CCC ACA GGG CAG TAA CGG CAC ACC

215

IVSI I (G-T)

TTA AAC CTG TCT TGT AAC CTT GAT ACG AAA

281

Fr4142
(TTCT)

GAG TGG ACA GAT CCC CAA AGG ACT CAA CCT

439

Del 619bP

CAA TGT ATC ATG CCT CTT TGC ACC

242

Cd15 (G-A )

TGA GGA GAA GTC TGC CGT TAC TGC CCA GTA

500

Cd5 (-CT)

ACA GGG CAG TAA CGG CAG ACT TCT CCG CGA

205

Cd 30 (G-C)

TAA ACC TGT CTT GTA ACC TTG ATA CCT ACG

280

Cd30 (G-A)

TAA ACC TGT CTT GTA ACC TTG ATA CCT ACT

280

Fr 16 (-C )

TCA CCA CCA ACT TCA TCC ACG TTC ACG TTC

238

Cd 26 (G-T)
(Hb- E)

TAA CCT TGA TAC CAA CCT GCC CAG GGC GTT

278

Cap +1 (A-C)

ATA AGT CAG GGC AGA GCC ATC TAT TGG TTC

567

Fr 47 48
(+ATCT)

ATA ACA GCA TCA GGA GTG GAC AGA TAG ATC

467

Hb-S

CCC ACA GGG CAG TAA CGG CAG ACT TCT GCA

207

1.Control

CAA TGT ATC ATG CCT CCT TGC ACC

2. Control

GAG TCA AGG CTG AGA GAT GCA GGA

3. Control

ACC TCA CCC TGT GGA GCC AC

4. Control

CCC CTT ATG ACA TGA ACT TAA

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Table-2.2 Primers used for identifying the normal alleles of the respective mutations.
Mutation

Primer

Used
with

Fragment
size

IVSI-5

CTC CTT AAA CCT GTC TTG TAA CCT TGT TAC

285

Fr 8 9

CCT TGC CCC ACA GGG CAG TAA CGG CAC ACT

214

IVSI I

GAT GAA GTT GGT GGT GAG GCC CTG GGT AGG

450

Fr4142

GAG TGG ACA GAT CCC CAA AGG ACT CAA AGA

443

Cd15

TGA GGA GAA GTC TGC CGT TAC TGC CCA GTG

500

Cd5

ACA GGG CAG TAA CGG CAG ACT TCT CCG CAG

207

Cd 30

TAA ACC TGT CTT GTA ACC TTG ATA CCT ACC

280

Fr 16

TCA CCA CCA TCA TCC ACG TTC ACG TTG

238

1.Control

CAA TGT ATC ATG CCT CTT TGC ACC

2. Control

GAG TCA AGG CTG AGA GAT GCA GGA

3. Control

ACC TCA CCC TGT GGA GCC AC

4. Control

CCC CTT CCT ATG ACA TGA ACT TAA

2.4.3: Electrophoresis :
Documentation of the amplified product was carried out on polyacrylamide gel
electrophoresis.

Preparing the gel:


Two glass sheets were washed with detergent and dried. Two sticks (3 mm thick) were
placed on the sides and the glasses were held together with clips. One side of the glasses

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was sealed with tape. 6% Acrylamide was prepared adding 28.5g of acrylamide (Merck),
1.5g Bisacrylamide (Fluka), 50ml of TBE (tris Boric acid Ethylenediametraaciticacid)and
500 ml of distilled water. Gel was prepared by adding 7 ml of 6% acrylamide, 50l of
ammonium per sulphate and 25l of TEMED (Invetrogen). The prepared mixer was then
poured in the glasses with the help of syringe, the comb was inserted and kept for 10 15
minutes till the gel sets. The comb was removed carefully. And the gel was loaded on the
electrophoresis chamber. The chamber was then filled with buffer. The buffer contains 1.8g
of tris (Life technology), Boric acid 5.5g, 0.9g of Na2EDTA (Merck) and distilled water
upto 1 liter. The well of the gel were cleaned and the mixture containing 4l of the
amplified product and 2l of the loading dye (Bromophenol blue) was loaded in each well.
The gel was run at 200 V for 35 minutes. 6l of an allelic ladder containing the mixture of
the amplified product of each of mutation was also added with each gel as control

Staining the gel:


The gel was stained with silver stain. The gel was removed from the chamber, one of the
glass sheets was removed carefully holding the gel on the other glass the gel was marked
and placed in a container. 1% Silver nitrate (BDH) was poured on the gel and kept for 10
minutes. After 10 minutes the stain was poured off. After giving a wash with water.
A mixture of 100ml sodium hydroxide(1.5%) and 75l of formaldehyde was poured. When
the bands were visible the mixture was poured off and gel was recovered on filter paper
and covered with cling wrap.

Multiplex ARMS PCR


As more than one mutation may be screened at the same time in a single PCR reaction
(Multiplex PCR), provided the ARMS primers are coupled with the same common primer

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(Tan et al.1994). The multiplex method of ARMS was accomplished by mixing several
primers in the same primer mixer. The mixing was organized according to the product size.
The combination of the primers used for screening in the multiplex PCR is presented in the
Table 2.3
The three groups of the multiplex primers were named AD-1, AD- 2 and AD- 3. The three
multiplex primers AD-I, AD 2 and AD 3 were prepared as stock solutions containing 5
pmol/ul each of all the primers for the mutant alleles. In addition, the two control primers
and respective common primer were also added to the stock mixture. In each ARMS
reaction 1 l of the stock primer was used.
Mutations can be easily identified by resolution on polyacrylamide gel electrophoresis
because the sizes of the amplified products are sufficiently different from each other. There
is no difference between IVSI-5 and Cd 30 (G - C and G A). IVSI I primer was added
to AD 1 and AD 2 groups, where as IVSI- 5 was added in AD 1. Codon 30 was added
in AD 2 and therefore amplification with AD-2 but not AD-1 indicated Cd 30. Same
primers were used for the detection of the homozygote and heterozygote for Cd30 and
IVSI-I. AD-3 includes the primers combination of Cd 15 and Cap +1. A control allelic
ladder was also prepared. Each fragment was amplified in 50 l reaction mixture under
standard ARMS condition. The individually amplified product were pooled and was used
as reference ladder.

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Table 2.3: Combination of various mutations screened by multiplex ARMS PCR


Primer ID

Mutations pooled

Amplified product size

AD-1

Fr 8 9(+G)
Del 619 bp
IVSI-1 (G-T)
IVSI-5 (G-C)
Fr 41 42

215bp
242bp
280bp
285bp
439bp

AD -2

Cd 5 (-CT)
Fr 16 (-C)
IVSI-1 (G-T)
Cd 30 (G-C)

205bp
238bp
280bp
280bp

AD-3

Cd15 (G-A)
Cap+1

500bp
567bp

10

11

12

control
bands

439bp
280bp
242bp
215bp

Fig 2.1: Polyacrylamide gel electrophoresis stained with silver stain. Sample 3 contains
Fr 8-9, sample 4 Del 619 and 5 Fr41-42. Sample 7 contains Cd5, sample 8 IVSI-I and 9
Fr16 mutation. Sample 11 contains Cap+1 and 12 Cd15 mutation. Sample 1 is ladder of
AD-1, 6 is ladder of AD-II and 10 is ladder of AD-III.

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RESTRICTION FRAGMENT LENGTH POLYMORPHISM


-Thalassemia mutations are known to create or abolish a restriction endonuclease site.
Majority of these can be detected quickly by restriction endonuclease analysis of
amplified DNA. The presence or absence of the enzyme recognition site is determined
from the pattern of digested fragments after agrose or polyacrylamide gel electrophoresis.
At least 18 restriction fragment length polymorphism (RFLPs) sites have been
characterized within the - globin gene cluster (Kazazian and Boehm 1986). -globin
gene cluster haplotype normally consist of five RFLPs. Located in the 5 cluster
(Table 2. 4)

Site

Enzyme
used

G gene

Hind - III

A gene

Hind III

5 Gene

Hind II

3 - gene

Hind II

- gene

Hinf I

- gene

Rsa I

Primer sequence:
5 AGT GCT GCA AGA ACA ACT ACC
5 CTC TGC ATC ATG GGC AGT GAG CTC
5- GAC TAG TGC TTG AAG GGG AAC AAC
5CCT CTG CTG ATT CAT TTC TTA CAC
5 TCC TAT CCA TTA CTG TTC CTT GAA
5ATT GTC TTA TTC TAG AGA CGA TTT
5 GTA CTC ATA CTT TAA GTC CTA ACT
5 TAA GCA AGA TA TTT CTG GTC TCT
5 TGG ATT CTG CCT AAT AAA A
5 GGG CCT ATG ATA GGG TAA T
5- AGA CAT AAT TTA TTA GCA TGC ATG
5 ACA TCA AGG GTC CCA TAG AC

Product
size
657
635
795

913

474
1200

Table 2.4: Polymorphic sites linked to the - globin gene, the restriction enzymes used
for their recognition, the sequence of the primers used for amplification of respective
fragments and the product sizes.

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Restriction endonuclease digestion of PCR product is a simple and reliable


technique for the diagnosis of point mutations, insertions or deletion which result in the
creation or abolition of a restriction endonuclease site. Primers are designed to span the
recognition site and produce easily identifiable fragments after digestion and
electrophoresis.
Xmn-I can recognize the C-T polymorphism at position 158 from the Cap site of the G
- globin gene (Thein et al , 1988). In order to demonstrate this polymorphism, a 641bp
fragment of DNA flanking the polymorphism was amplified using the following primers.

5 GAA CTT AAG AGA TAA TGG CCT AA


5 ATG ACC CAT GGC GTC TGG ACT AG

The reaction mixture was prepared by adding 20 l of buffer, 1 l of primer, 2 l of DNA


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and 0.1 l of Taq. The PCR conditions were same as for the RFLP protocol with 94 C for
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1 minute denaturation time, Annealing at 60 C for 1 minute extension at 72 C for 1


o

minute and 3 minutes of final extension at 72 C. Numbers of cycles carried out were 30.
Tubes were removed from the PCR machine and the amplified fragment was digested
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with 10 units of enzyme Pdm-I (Fermentus) at 37 C overnight and the results were
recorded after electrophoresis on 6% Acrylamide gel.

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2.5: CHARACTERIZATION OF - THALASSEMIA


- Thalassemia can be diagnosed by the identification of characteristic abnormal DNA
fragments which span the breakpoint of the deletion.
The buffer mixture taken was 20 l to which 1 l of primer and 0.1 l of taq was added.
2 l of DNA was added to each mixture tube.

641 bp
418bp

223bp
1

10

Fig 2.2: Silver stained PAGE of the PdmI (XmnI ) digested PCR products. The 641-bp
represents the uncut (-) site, while the 418-bp and 223-bp fragments represent the cut
(+)site. Samples 1,2,6, 7, 8,9 and 10 are +/+ , where as sample 4 and 5 are -/+ , and
sample 3 is -/-.

641 bp
418bp

223bp

Fig 2.3: Silver stained PAGE of the PdmI (XmnI ) digested PCR products. The 641-bp
represents the uncut (-) site, while the 418-bp and 223-bp fragments represent the cut (+)
site. Samples 1,3,5 and 6 are -/+ , where as sample 7 is +/+ , and 2,4 and 8 are -/-.

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In the PCR reaction the denaturation was carried out at 94oC for 1 minute , annealing
o

temperature was 60oC for 1 minute, extension at 72 C for 1 minute and a final extension
was carried out at 72oC for 3 minutes. The amplification was carried out by 30 cycles.
The amplified product was run on 6% acrylamide gel and the results were recorded.

2.6: Gene Analysis For Thalassemia


1 and 2 genes are embedded with two highly homologous 4-kb duplicated segments.
They are further subdivided into X,Y and Z separated by the nonhomologous regions I,II
and III. The chromosome with a hybrid 2/1 gene (-3.7 deletion) results from the
reciprocal recombination between Z segments which are 3.7 kb apart. Whereas
recombination between homologous X segments which are 4.2 kb apart, result in a single
1 gene, thus deleting the entire 2 gene (-4.2 deletion).

PCR conditions for the detection 3.7 and 4.2 deletion


The common -thalassemia -2 determinants can be detected by PCR (Baysal and
Huisman 1994). Appropriate segments of the chromosome with the deletions and the
normal chromosome can be selectively amplified with PCR primers under identical
experimental conditions. The products are different in size and can be separated by
Acrylamide gel electrophoresis.
Specific primers were designed to allow the selective amplification of the 2 and
1genes. Their sequences are given in the table 2.5.

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Table : 2.5: Primers used for detection of thalassemia deletions (Baysal and Huisman
1994)
Primer ID

Sequence

Concentration
used

A:

CTT TCC CTA CCC AGA GCC AGG TT

25pmol/reaction

B:

CCC ATG CTG GCA CGT TTC TGA GG

C:

CCA TTG TTG GCA CAT TC GGG ACA

D:

CCT TCC TCT CAC TTG GCC CTG AG

E:

CCC TGG GTG TCC AGG AGC AAG CC

F:

CGC CTC CCT GGA CAA GTT

G:

CCG GTT TAC CCA TGT GGT GCC TC

25pmol/reaction
25pmol/reaction
20pmol/reaction
15pmol/reaction
15pmol/reaction
15pmol/reaction

The location of the amplification primers flanking the deletion breakpoints. To


3.7
demonstrate the presence of the - genetype, two separate reactions were run

simultaneously for each DNA sample. One reaction contains the pair of primers
that amplified a segment of the chromosome with the deletion consisting of
sequence 5 and 3 to the deletion (A+B), while the other pair (A+C) amplified a
comparable segment of the normal gene (fig 2.4).
A similar strategy was used to test the presence of - 4.2 genotype. Primers D+E or

+E will amplify the segment of the abnormal chromosome with the - 4.2 deletion,

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whereas D + F or G + F will amplify that of the normal gene. The design of the reverse
primers B and C is based on the sequence divergence between the 1 and 2 genes,
immediately 3 to the poly A site.
Gap PCR can be used for the detection of - chain deletions. The PCR buffer was
prepared by mixing 67 mM Tris Hcl , Ph 8.8; 16.6mM ammonium sulphate, 0.10mg/ml
Bovine serum Albumin , 10 mM -Mercaptoethanol, 4.0 mM Mgcl2 , 10% DMSO, 200
mM dNTPs and 2.0 units of Taq polymerase (Perkin Elmer). Detection of normal and
mutant allele for - 3.7 was carried out by performing PCR in two separate tubes because
the amplified product under both conditions is of the same size and can not be separated
on electrophoresis. PCR reactions for - 4.2 were carried out in the same tube because of
different product size.
PCR was carried out with Hot start method i.e the taq polymerase was added when the
reaction mixture was above 800C. The PCR conditions were; denaturation at 940C for 1
minute, primer annealing at 60oC for 1 minute and extension for 3 minutes at 72oC.
Extension in the last cycle was prolonged for further 8 minutes. The amplified product
was separated on 6% acrylamide gel.

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B
3.7kb

-3.7deletion

-4.2deletion
4.2kb

D
I

X2
B
-

II

Y2
3.7

Z2

X1
Y2

III

Y1

Z1

Z2 - 1

hybrid gene
A

B
A pa I site

Type I: 1.5 kb
Type I I: 1.8 kb

Fig. 2.4: A: Strategy for the amplification of segments of DNA from chromosomes with
the two types of - thal 2 deletion. The functional 2, 1 and 1 genes are shown as
open boxes. The solid boxes indicated the extend of the deletions involving the 1 and
2 genes. Also indicated are the three homologous boxes X,Y,Z and the nonhomologous
regions I , II and III. The locations of the primers are indicated by the vertical lines and
their directions by the arrows. For the - 3.7 detection, primers A + B will amplify the
abnormal segment and A + C will amplify the normal sequence. A similar approach is
used for the detection of the - 4.2 with primers D + E and D + F or with G + E and
G + F. Primer pairs A + B, D+ E and G + E will not yield any product form the normal
chromosome since they are located too far apart (>7kb). B: Identification of the - 3.7
genotype, type I and II. The locations of primers A and B are shown by horizontal arrows
in the Z2 -1 homologous region, which is specific for the - 3.7 hybrid gene. The Apa1
restriction site within a 7-bp insert in the IVS-II of the 1 gene is present in type I, but
not in type II, because of different crossover events. The horizontal lines show the sizes
of the expected fragments after digestion of the amplification product with the Apal
enzyme (Baysal and Huisman 1994).

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