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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
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
2. Control
3. Control
4. Control
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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
238
1.Control
2. Control
3. Control
4. Control
2.4.3: Electrophoresis :
Documentation of the amplified product was carried out on polyacrylamide gel
electrophoresis.
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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
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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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Mutations pooled
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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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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and 0.1 l of Taq. The PCR conditions were same as for the RFLP protocol with 94 C for
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
o
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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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.
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Table : 2.5: Primers used for detection of thalassemia deletions (Baysal and Huisman
1994)
Primer ID
Sequence
Concentration
used
A:
25pmol/reaction
B:
C:
D:
E:
F:
G:
25pmol/reaction
25pmol/reaction
20pmol/reaction
15pmol/reaction
15pmol/reaction
15pmol/reaction
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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