Gap PCR

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Electronic Infrastructure
For Thalassaemia Research Network

PROTOCOL
Gap - PCR

Gap-PCR

Principle
Gene deletion mutations in the -globin gene cluster may be detected by PCR using
two primers complimentary to the sense and antisense strand in the DNA regions that
flank the deletion. For small deletions of less than one kilobase, the primer pair will
generate two products, the smaller fragment arising from the deletion allele. For large
deletions, the distance between the two flanking primers is too great to amplify the
normal allele and product is only obtained from the deletion allele. In these cases the
normal allele is detected by amplifying across one of the breakpoints, using a primer
complimentary to the deleted sequence and one complimentary to the flanking DNA.

Gap-PCR is used for to diagnose some -thlassaemia deletions, HPFH deletions, -
thalassaemia deletions (Table 5.2) and also the triple a-gene locus generated by the
3.7kb single -gene deletion [5]. A typical gap-PCR test is illustrated in Figure 5.10
and 5.11. For the diagnosis of -thalassaemia, the primers can now be multiplexed.
The 3.7kb and 4.2kb
+
-thalassaemia deletions can be detected in one assay (table
5.9), the --
MED
and -()
20.5

-thalassaemia deletions in one assay (table 5.10) and the

3 Southeast Asian
-thalassaemia deletions in one assay (Table 5.11). The protocols

used in the Oxford laboratory for the multiplexing of these primers are given in
Tables 5.9-5.11, but it should be noted that the quantity of each primer pair relative to
the others may need adjustment to gain optimum amplification of all the products.
PCR diagnosis of the triple -gene (anti 3.7 allele) requires two separate assays
section
Molecular diagnosis procedures Diagnosis of known mutations

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(Tables 5.12 & 5.13). The presence of the allele is diagnosed from a comparison of
the results of each assay run side by side and the genotype of the DNA sample can be
deduced (Table 5.14) in most instances.

Table 5.2. Thalassaemia deletion mutations which have been diagnosed by gap-PCR

o
-thalassaemia

+
-thalassaemia

o
-thalassaemia

()
o
-thalassaemia

(
A
)
o
-thalassaemia

HPFH

--
SEA

--
MED

-()
20.5

--
FIL
--
THAI

-
3.7

-
4.2

290 bp deletion
532 bp deletion
619 bp deletion
1393 bp deletion
1605 bp deletion
3.5 kb deletion
10.3 kb deletion
45 kb deletion

Hb Lepore
Spanish
Sicilian
Vietnamese
Macedonian/Turkish

Indian
Chinese

HPFH1 (African)
HPFH2 (Ghanaian)
HPFH3 (Indian)
Hb Kenya

[6]
[6]
[6]
[8,9]
[8.9]

[7]
[7]

[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]

[20]
[20]
[20]
[20]
[20]

[20]
[20]

[20]
[20]
[20]

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Method
1. Set up the reaction mixture to a final volume of 22 l into a 0.5 ml tube with
the following components as required: 1 l genomic DNA (100 ng/l) (1 l of
forward primer - flanking sequence (10 pmol/l), 1 l reverse primer -
flanking sequence (10 pmol/l),1 l of primer - deleted sequence (10
pmol/l), 1 l of primer - inverted sequence (10 pmol/l), 2.5 l of 1.25 mM
(dNTP mixture), 2.3 l of 10x Gap PCR buffer as recommended for the
primers in the original reference (see Table 5.2) and below.
2. The buffer recommended for the -thalassaemia primers is 750 mM Tris-HCl
pH 8.8, 200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20). The buffer for the -
thalassaemia primers should also contains 0.5 M betaine and 0.5% DMSO
(this can be achieved by adding 2.5 l of 5 M betaine and 1.25 l 10%
DMSO).
3. Make up all reactions to a final volume of 22 l by adding sterile dH
2
O.
4. Overlay with 25 l of mineral oil.
5. Prepare enzyme mixture: 0.2 l reaction buffer (10x), 0.1 l AmpliTaq
(5U/l) (PE Biosystems) for the -gene primers, 0.1 l Platinum Taq (5U/l)
(Invitrogen) for the -gene primers, and 2.7 l sterile dH
2
O, to make 3 l.
6. Mix enzyme mixture and hold on ice.
7. Place reaction mixtures in thermal cycler and perform one cycle as follows,
adding 3 l of the enzyme mix after 2 minutes of the 94
o
C denaturation step:
4 min at 94
o
C/ 1 min at 55-65
o
C (as recommended)/ 1.5 min at 72
o
C
8. Continue for 33 cycles with the following steps per cycle: 1 min at 94
o
C/1
min at 55-65
o
C (as recommended in the published references or in tables 5.9-
5.13)/1.5 min at 72
o
C
9. Finish with one cycle as follow: 1 min at 94
o
C/1 min at 55-65
o
C (as
recommended)/10 min at 72
o
C.
10. Hold at 15
o
C until gel electrophoresis.
11. Remove tubes from thermal cycler. Add 5 l of blue dye, mix and centrifuge.
12. Depending on expected product sizes, load a 20 l aliquot onto a 1-3%
agarose gel and run at 100 V for 45 min to 2 hrs in 1x Tris-borate-EDTA
buffer (TBE).

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13. Stain gel in ethidium bromide solution (0.5 g/ml) for 15-30 minutes,
visualise bands on a UV light box (312 nm) and photograph with an electronic
camera system or a Polaroid CU-5 camera fitted with an orange filter (e.g.
Wratten 22A). For guidance re interpretation see notes 6,7 and 8.

Materials
a) DNTPs: Add together 50 l of a 100 mM solution of each dNTP (as
purchased) and 3.8 ml of distilled water. The 1.25 mM dNTP stock solution
should be stored in frozen aliquots.
b) 10x Gap PCR reaction buffer (composition varies according to primers used)
see methods and Table 2
c) Betaine (Sigma-Aldrich Chemical Co Ltd, England)
d) Mineral Oil to overlay PCR reactions
e) PCR primers: dilute aliquots of primer stock solutions to make a working
solution of 1 OD unit/ml and store frozen.
f) Ammonium sulphate buffer: 75 mM Tris-HCl (pH 9.0), 20 mM (NH
4
)
2
SO
4
,
2.0 mM MgCl
2
, 0.01% Tween 20, 10% DMSO, 10 mM -mercaptoethanol
(all final concentrations).
g) Taq polymerases and 10x Taq buffers: in my laboratory are as follows,
AmpliTaq Gold (PE Biosystems) works best for ARMS-PCR/RE digestion
assays and Platinum Taq (Gibco Life Technologies) for gap-PCR.
h) Tris-borate -EDTA (TBE) buffer : 89 mM Tris-borate, 89 mM boric acid, 10
mM EDTA, pH 8.0.
i) Blue running dye (15% ficoll/0.05% bromophenol blue).
j) UV transilluminator and Polaroid camera, or UV electronic camera system
k) 0.5 g/l Ethidium bromide

Multiplex Gap-PCR
Specific primer details etc are listed below for the multiplex diagnosis of the common
-thalassaemia genotypes and the triplicated -globin allele. Figures 5.10 and 5.11
show example results all the common -thalassaemia geneotypes.

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1. Multiplex PCR protocol for the diagnosis of
3.7
and
4.2
deletions.

A) Primer sequences
primer description sequence Annealing temp
0
C
1 2/3.7-F CCCCTCGCCAAGTCCACCC 64
2 3.7/20.5-R AAAGCACTCTAGGGTCCAGCG 64
3 2-R AGACCAGGAAGGGCCGGTG 64
4 4.2-R CCCGTTGGATCTTCTCATTTCCC 64
5 4.2-F GGTTTACCCATGTGGTGCCTC 64

B) PCR reaction mix
component
l
2/3.7-F (10 M)
1.0
2-R (10 M)
0.25
2/20.5-R (10 M)
1.0
4.2-F (10 M)
1.0
4.2-R (10 M)
1.5
10x buffer (750 mM Tris-HCl pH 8.8,
200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20)
2.5
25 mM MgCl
2

1.5
dNTPs (1 mM)
5.0
Betaine (5 M)
3.75
DMSO (10%)
1.25
Platinum Taq (5 units /l)
0.1
DNA template (100 ng/l)
1.0
Water
5.2

C) Gel electrophoresis conditions
Run PCR products out on 1.5% (1:1 Nusieve:agarose ) gel for 2-3 hours

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D) Interpretation of results
PCR Fragment size (bp) Genotype Product of primers
2020
+
-thalassaemia: -
3.7
1 + 2
1800 Normal () 1 + 3
1628
+
-thalassaemia: -
4.2
4 + 5

2. Multiplex PCR protocol for the diagnosis of the --
MED
and -()
20.5
deletions.

A) Primer sequences
primer name sequence Annealing
temp
0
C
1 MED(F) CGATGAGAACATAGTGAGCAGAATTGCAGG 60
2 MED(R) ACGCCGACGTTGCTGCCCAGCTTCTTCCAC 60
3 SEA(F) CTCTGTGTTCTCAGTATTGGAGGGAAGGAG 60
4 SEA(N) TGAAGAGCCTGCAGGACCAGGTCAGTGACCG 60
5 -()
20.5
(F) GGGCAAGCTGGTGGTGTTACACAGCAACTC 60
6 -()
20.5
(R)
CCACGCCCATGCCTGGCACGTTTGCTGACG 60

B) PCR reaction mix
component l
SEA(F) (10 M) 1.0
SEA(N) (10 M) 0.5
MED(F) (10 M) 0.4
MED(R) (10 M) 0.4
-()
20.5
(F) (10 M) 0.4
-()
20.5
(R) (10 M) 0.4
200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20)
2.5
25 mM MgCl
2
1.5
dNTPs (1 mM) 4.0
Betaine (5 M) 3.75
DMSO (10%) 1.25
Platinum Taq (5 units /l) 0.1
DNA template (100 ng/l) 1.0
Water 6.2

Run PCR products out on 2% (1:1 Nusieve:agarose ) gel for 1-1.5 hours

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1175
0
-thalassaemia: -()
20.5
5 + 6
1010 Normal () 3 + 4
875
+
-thalassaemia: --
MED
1 + 2

3. Multiplex PCR protocol for the diagnosis of the --
SEA
/ --
FIL
/ --
THAI

0
-
thalassaemia deletions.

A) Primer sequences
primer name Sequence Annealing
temp
0
C
1 FIL (F) AAGAGAATAAACCACCCAATTTTTAAATGGGCA 60
2 FIL (R) GAGATAATAACCTTTATCTGCCACATGTAGCAA 60
3 SEA(F) CTCTGTGTTCTCAGTATTGGAGGGAAGGAG 60
4 SEA(N) TGAAGAGCCTGCAGGACCAGGTCAGTGACCG 60
5 SEA(R) ATATATGGGTCTGGAAGTGTATCCCTCCCA 60
6 THAI(F) CACGAGTAAAACATCAAGTACACTCCAGCC 60
7 THAI(R) TGGATCTGCACCTCTGGGTAGGTTCTCTACC 60

B) PCR reaction mix
component l
SEA(F) (10 M) 2.0
SEA(N) (10 M) 1.0
SEA(R) (10 M) 1.0
FIL (F) (10 M) 4.0
FIL (R) (10 M) 4.0
THAI (F) (10 M) 1.0
THAI (R) (10 M) 1.0
200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20)
2.5
25 mM MgCl
2
1.5
dNTPs (1 mM) 4.0
Betaine (5 M) 3.75
DMSO (10%) 1.25
Water 0.65

Run PCR products out on 1.5% (1:1 Nusieve:agarose ) gel for 2 hours

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1010 Normal () 3 + 4
660
+
-thalassaemia: --
SEA
3 + 5
550
+
-thalassaemia: --
FIL
1 + 2
495
+
-thalassaemia: --
THAI
6 + 7

4.1 PCR protocol for the diagnosis of the (anti 3.7) allele: reaction mix 1

A) Primer sequences
primer description sequence Annealing
temp
0
C
1 C10 GATGCACCCACTGGACTCCT 55
2 C3 CCATTGTTGGCACATTCCGG 55

B) PCR reaction mix
component l
C10 (10 M) 1.0
C3 (10 M) 1.0
200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20)
2.5
25 mM MgCl
2
1.5
dNTPs (1 mM) 5.0
Betaine (5 M) 3.75
DMSO (10%) 1.25
Water 12.9

Run PCR products of reaction mixture 1 out on 2% (1:1 Nusieve:agarose ) gel for 2
hours, in lane next to those of reaction mixture 2. See Table 5.14 for interpretation of
results.

D) Product sizes
No product
+
-thalassaemia: -
3.7
1 + 2
1900 Normal () 1 + 2
1900 : (anti 3.7) 1 + 2

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4.2 PCR protocol for the diagnosis of the (anti 3.7) allele: reaction mix 2

E) Primer sequences
primer Description sequence Annealing temp
0
C
1 C10 GATGCACCCACTGGACTCCT 50
2 C2 CCATGCTGGCACGTTTCTGA 50

F) PCR reaction mix
Component l
C10 (10 M) 1.0
C2 (10 M) 1.0
200 mM (NH
4
)
2
SO
4
, 0.1% Tween 20)
2.5
25 mM MgCl
2
1.5
dNTPs (1 mM) 5.0
Betaine (5 M) 3.75
DMSO (10%) 1.25
Water 12.9

G) Gel electrophoresis conditions
Run PCR products of reaction mixture 2 out on 2% (1:1 Nusieve:agarose ) gel for 2
hours, in lane next to those of reaction mixture 1. See Table 5.14 for interpretation of
results.

H) Product sizes
2100 Normal () 1 + 2
2100 : (anti 3.7) 1 + 2
1900
+
-thalassaemia: -
3.7
1 + 2

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4.3 Interpretation of the results of reaction mixes 1& 2 for the diagnosis of the
(anti 3.7) allele.

A) Products (bp)

Genotypes

Primers: C2+C10

Primers: C3+C10

/ 2100 1900
-
3.7
/ 2100 + 1900 1900
-
3.7
/ -
3.7
1900
/ -
3.7
2100 + 1900 2100 + 1900
/ or / 2100 2100 + 1900

Notes:
i. allele: amplifies with C3+C10 (1.9kb) and C2+C10 (2.1kb).
ii. The -
3.7
allele: amplifies with only C2+C10. Gives a shorter
product (1.9kb) than normal because of the deleted -gene.
iii. The allele: amplifies with C3+C10 (1.9kb) and twice with
C2+C10 (2.1kb) because of the extra -gene.

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B) Possible gel patterns

-genotypes

/ -
3.7
/ -
3.7
/ -
3.7
/ -
3.7
/ or
/

Primer pairs

C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C0

Band patterns

____

____

____

____

____

____

2.1 kb
____ ____ ____ ____

____ ____ ____

1.9 kb

Figure 5.10.
The diagnosis of
+
-thalassaemia deletion mutations by multiplex GAP PCR using
the primers described in Table 5.9.

H
i
n
d

I
I
I

H
i
n
d

I
I
I
X
1
7
4
X
1
7
4
3
.
7
/
3
.
7
/

3
.
7
3
.
7
/

4
.
2
4
.
2
/
3
.
7
/

4
.
2
4
.
2
/

4
.
2

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Figure 5.11
The diagnosis of
0
-thalassaemia deletion mutations by multiplex GAP PCR using the
primers described in Tables 5.10 & 5.11.
X
1
7
4
X
1
7
4
S
E
A
/
S
E
A
/

S
E
A
T
H
A
I
/
F
I
L
/
M
E
D
/
)
2
0
.
5
/

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-thalassaemia deletions in one assay (table 5.10) and the

-thalassaemia deletions in one assay (Table 5.11). The protocols

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