Ipsogen JAK2 MutaQuant Kit Handbook

2013
ipsogen JAK2
MutaQuant
12 (. 673522)
24 (. 673523)
1
in vitro
Rotor-Gene Q, ABI PRISM
7900HT SDS, Applied Biosystems 7500 Real-Time PCR
LightCycler
673522, 673523
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden,
R1
1072501EL
Sample & Assay Technologies
QIAGEN: Sample and Assay Technologies

QIAGEN
,
. ,
-
.
QIAGEN :
DNA, RNA
microRNA RNAi
.
,
www.qiagen.com.
11
11
12
13
14
15
16
DNA
16
qPCR RotorGene Q MDx 5plex HRM Rotor
17
qPCR ABI PRISM 7900HT SDS, Applied Biosystems 7500

Real-Time PCR, LightCycler 480
22
qPCR LightCycler 1.2

29
34
38
42
42
42
42
44
45
46
47
48
ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant in vitro
JAK2
V617F/G1849T DNA
(MPN).
JAK2 V617F/G1849T
JAK2.

88504 88622 (1).
:
,
.
/
QIAGEN.

, V617F,
Janus 2 (JAK2), 2005 (25),
,
(MPN). JAK2
,
.
JAK2 V617F >95%
(polycythemia vera, PV), 5060%
(essential thrombocythemia, ET) 50%
(primary myelofibrosis, PMF). JAK2
V617F
, ,
,
0% (6).
, JAK2
1849 14,
(V) (F) 617
( JH2). JAK2,
in vitro,
(EEC) PV
ET PMF (7). JAK2 V617F

MPN, ,
,
, .
, MPN ,
.
4

.
JAK2 V617F .. 2008
BCR-ABL MPN ( 1),
.
1. ... MPN (
8)
(PV)
1. (Hgb) >18,5 g.dl-1 () >16,5 g.dl-1

()
Hgb (Hct) >99
,
Hgb >17 g.dl-1 () >15 g.dl-1 ()
2 g.dl-1
,

>25%

2. JAK2V617F
1.
2.
3. (EEC)
(ET)
1. 450 x 109 l-1

2.
.

3. ...
(, CML), PV,
(PMF), (,
MDS),
4. JAK2V617F

(PMF)
1.
/
,

,
(.
PMF)
2. ... , PV,

3. JAK2V617F

(PMF)
1.
2. (LDH)
3.
4.
,
PV ET. ,
- , JAK2V617F

(9). JAK2 V617F
JAK2 (10).

(SNP)
DNA. ,
(qPCR)
,
.
110%, ,
TaqMan, Pyrosequencing (),
, . ,
, -, ,
, PCR

.
<0,1% SNP (primer)

( ) ,
qPCR . ipsogen JAK2
MutaQuant .
qPCR PCR
PCR.
PCR ,
PCR,
/ PCR,
PCR. ,
3 qPCR: qPCR
SYBR Green I, qPCR
, qPCR
.

qPCR. PCR,
.
.
,
(reporter) 5'
(quencher) 3' ,
- PCR. qPCR
5'3'
DNA Thermus aquaticus (Taq).
,

Frster.
PCR, ,

. 5'3' DNA

.
,
. 3'
PCR (
1).
.
-

PCR. ,
. ,
PCR.

PCR

PCR
Taq
1. .
PCR
,
SNP.
, V617F
(11). DNA-
PCR (. 2).
WT
WT
2. PCR.
V617F

.
VF JAK2.
10

ipsogen JAK2 MutaQuant Kit
.

(12)
(24)
673522
673523
12
24
V617F positive control (

V617F)
(100% V617F)
PC-VF-JAK2
PC-VF-JAK2
40 l
60 l
V617F negative control (

V617F)
(100%
)
NC-VF-JAK2
NC-VF-JAK2
40 l
60 l
M1-VF Standard Dilution (

M1-VF),
50
(5 x 101 V617F /5 l)
M1-VF
M1-VF
20 l
30 l

M2-VF),
500 (5 x 102 V617F
/5 l)
M2-VF
M2-VF
20 l
30 l

M3-VF),
5.000 (5 x 103 V617F
/5 l)
M3-VF
M3-VF
20 l
30 l

M4-VF),
50.000 (5 x 104 V617F
/5 l)
M4-VF
M4-VF
20 l
30 l
WT-1 Standard Dilution (

WT-1),
50
(5 x 101
/5 l)
WT-1
WT-1
20 l
30 l

WT-2),
500
(5 x 102
/5 l)
WT-2
WT-2
20 l
30 l
11
ipsogen JAK2 MutaQuant Kit

.

(12)
(24)
673522
673523
12
24
20 l
30 l
20 l
30 l

WT-3),
5.000 (5 x 103
/5 l)
WT-3
WT-3

WT-4),
50.000 (5 x 104

/5 l)
WT-4
WT-4
Primers and Probe Mix JAK2 WT

(
JAK2 WT)*
PPM-JAK2 WT 25x
PPM-JAK2 WT 25x
52 l
95 l
Primers and Probe Mix JAK2 V617F PPM-JAK2 V617F 25x

(
PPM-JAK2 V617F
JAK2 V617F)
25x
52 l
95 l
ipsogen JAK2 MutaQuant Kit Handbook ()
* JAK2 FAMTAMRA.
JAK2 V617F
FAMTAMRA.
:
.

, .
, (SDS),
.
PCR
Taq DNA :
TaqMan Universal PCR Master Mix (
PCR 2x) (Life Technologies, 4304437) LightCycler
TaqMan Master ( PCR 5x) (Roche,
04535286001) LightCycler FastStart DNA MasterPLUS HybProbe (
5x) (Roche, 03515567001)
12
PCR

PCR 0,5 ml 1,5 ml
* PCR (110 l, 10100 l,

1001.000 l)
*
0,5 ml/1,5 ml (
13.00014.000 rpm)
PCR :* Rotor-Gene Q 5plex HRM

Rotor Gene, LightCycler 1.2, 2.0 480, ABI PRISM 7000, 7700
7900HT SDS, SmartCycler,

in vitro

, .

(SDS). online
PDF www.qiagen.com/safety ,
SDS
QIAGEN.

.
*
.
13
, 24
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24 :
(Poison Information Center) Mainz
.: +49-6131-19240

qPCR ,
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16
: qPCR RotorGene Q MDx 5plex

HRM Rotor
,
, 2.
2. Rotor-Gene Q
72
JAK2 V617F (PPM-VF)

4 M-VF
8 ,

n DNA
n x 2
2 DNA
4 :
(PC-VF)
(NC-VF),
JAK2
(PPM-WT)
4
8 ,

n DNA
n x 2
2 DNA
4 : PC-VF NC-VF,

Rotor-Gene Q 72
8 DNA 24
(. 673523) 6 DNA
12 (. 673522) ,

.
17
3. ipsogen
JAK2 MutaQuant 24 . PC-VF: V617F, NC-VF:
V617F, M-VF: V617F, M-WT: , S:
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:
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.
qPCR Rotor-Gene Q 72
: .
1.
.
18
2. qPCR
.
.
3 4
,
25 l.
-, ,
( PPM-VF PPM-WT).

.
3. qPCR
1
(l)
-
V617F
30 + 1
(l)

TaqMan Universal
PCR, 2x
12,5
387,5
1x

, PPM-VF
25x
1,0
31
1x

PCR

6,5
201,5
(

4)
5,0
25,0
25
19
4. qPCR
1
(l)
- WT
30 + 1
(l)

TaqMan Universal
PCR, 2x
12,5
387,5
1x

, PPMWT
25x
1,0
31
1x

PCR

6,5
201,5
(

4)
5,0
25,0
25
3. 20 l - qPCR (VF WT) .

4. 5 l (25 ng
DNA )
( 25 l).
5. , /
.
6.
.
7. Rotor-Gene Q
, 5.
20
5.
: 50C
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: 10
50
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62C 1 FAM
Green:
8. Rotor-Gene Q, Slope Correct (

) .
0,03. ,
5.
21
: qPCR ABI PRISM 7900HT SDS,

Applied Biosystems 7500 Real-Time PCR,
LightCycler 480
qPCR 96 ,
,
6.
6. qPCR
96
JAK2 V617F (PPM-VF)

4 M-VF
8 ,

n DNA
n x 2
2 DNA
4 : PC-VF NC-VF,

JAK2
(PPM-WT)
4
8 ,

n DNA
n x 2
2 DNA
4 : PC-VF NC-VF,

ABI PRISM 7900HT SDS, Applied

Biosystems 7500 Real-Time PCR, LightCycler 480
8 DNA 24 (.
673523) 6 DNA 12
(. 673522) ,

.
4
24 (. 673523),
22
5
12 (. 673522).
4.
24 (. 673523). PC-VF: V617F, NC-VF:
V617F, M-VF: V617F, M-WT:
, S: DNA, H2O:
23
5.
12 (. 673522). PC-VF: V617F, NC-VF:
V617F, M-VF: V617F, M-WT:
, S: DNA, H2O:
qPCR ABI PRISM 7900HT SDS, Applied Biosystems 7500

Real-Time PCR, LightCycler 480
: .
1.
.
2. qPCR
.
.
7 8
,
25 l.
-, ,
( PPM-VF PPM-WT).

.
24
7. qPCR
- V617F
1
(l)
26 + 1
(l)
30 + 1
(l)
TaqMan
Universal
PCR, 2x
12,5
337,5
387,5
1x

,
PPM-VF
25x
1,0
27
31
1x
PCR
6,5
175,5
201,5

4)
5,0
25,0
25
25
25
8. qPCR
- WT
1
(l)
26 + 1
(l)
30 + 1
(l)
TaqMan
Universal
PCR, 2x
12,5
337,5
387,5
1x

,
PPM-WT
25x
1,0
27
31
1x
PCR
6,5
175,5
201,5

4)
5,0
25,0
25
25
3. 20 l - qPCR (VF WT) .

4. 5 l (25 ng
DNA )
( 25 l).
5. , /
.
6. (300 x g,
10 ).
7.
.
8.

FAM ,
26
9 ABI PRISM 7900HT SDS Applied

Biosystems 7500 Real-Time PCR, 10
LightCycler 480.
9. ABI PRISM 7900HT
SDS Applied Biosystems 7500 Real-Time PCR
: 50
: 2
: 95
: 10
50
95C 15
63C 1 30
FAM, :
TAMRA
10. LightCycler 480
(Abs Quant)
Simple Probe ( )
Detection formats ( )
: 50C
: 2
: 95C
: 10
50
95C 15
63C 1 30
FAM (483533 nm)
LC 01 (465510 nm) LC
02
27
9. ABI PRISM 7900HT Applied Biosystems 7500

Real-Time PCR, 8. LightCycler
480, 8.
9. ABI PRISM 7900HT Applied Biosystems 7500 Real-Time
PCR: 0,1.
, 9.
9. LightCycler 480: Fit point
( ) 2,0 2,0.
,
10.
28
: qPCR LightCycler 1.2

,
,
11.
11. LightCycler 1.2
JAK2 V617F (PPM-VF)

4 M-VF
4 ,

n DNA
n x 2
2 DNA
2 : PC-VF NC-VF,

JAK2
(PPM-WT)
4
4 ,

n DNA
n x 2
2 DNA
2 : PC-VF NC-VF,

LightCycler 1.2
4 DNA

. 6
.
29
6. ipsogen
JAK2 MutaQuant. PC-VF: V617F, NC-VF: V617F,
M-VF: V617F, M-WT: , S: DNA, H2O:
.
qPCR LightCycler 1.2

: ,
LightCycler
. LightCycler FastStart DNA
MasterPLUS HybProbe
5x.
: .
1.
.
2. qPCR
.
.
12 13
,
20 l.
-, ,
( PPM-VF PPM-WT).

.
30
12. qPCR
1
(l)
-
V617F
15 + 1
(l)
LightCycler
FastStart DNA
MasterPLUS
HybProbe, 5x
4,0
64,0
1x

, PPM-VF
25x
0,8
12,8
1x

PCR

10,2
163,2
(

4)
5,0
20,0
20
31
13. qPCR
1
(l)
-
WT
15 + 1
(l)
LightCycler
FastStart DNA
MasterPLUS
HybProbe, 5x
4,0
64,0
1x

, PPMWT 25x
0,8
12,8
1x

PCR

10,2
163,2
(

4)
5,0
20,0
20
3. 15 l - qPCR (VF WT) .

4. 5 l (25 ng
DNA )
( 20 l).
5. , /
.
6.
, (700 x g, 10
).
7.
.
8. LightCycler 1.2
, 14.
32
14.
: 55
: 2
: 20
: 95
: 10
: 20
50
95C 15 , : 20
66C 1 , : 20,
FAM:
9. LightCycler 1.2, F1/F2 2nd

derivative analysis ( 2 ).
,
14.
33

(CT)
(CP) qPCR
Excel .
CP CT
CT

V617F 15.
y = CP, x = log10 CN CN=
5 l
15.
V617F

(CN)
log10 CN
M1-VF
5 x 101 VF
1,7
M2-VF
5 x 102 VF
2,7
M3-VF
5 x 103 VF
3,7
M4-VF
5 x 104 VF
4,7
WT-1
5 x 101 WT
1,7
WT-2
5 x 102 WT
2,7
WT-3
5 x 103 WT
3,7
WT-4
5 x 104 WT
4,7
:
.

7 9
ipsogen JAK2 MutaQuant 8 10

4 .
34
M4-VF M3-VF M2-VF
M1-VF
7. 5 x 101, 5 x 102, 5 x 103 5 x 104

JAK2 V617F ( M1-VF, M2-VF, M3-VF, M4-VF, ).
y = -3,4017x + 44,774
R = 0,9989
8. JAK2 V617F.
35
WT-4 WT-3 WT-2 WT-1
9. 5 x 101, 5 x 102, 5 x 103 5 x 104

JAK2 ( WT-1, WT-2, WT-3 WT-4,
).
y = -3,6x + 43,643
R = 0,9981
10. JAK2 .
10- ,
3,32. 3,0 3,9
36
R >0,95 (12). , R >0,98

(13).

V617F WT log10
.
V617F
CP/ CT (
PPM-VF) ,
JAK2 V617F (CNV617F).
log10 CNV617F =
( CpV617F
V617F)
V617F

CP/CT (
PPM-WT) ,
JAK2 (CNWT).
log10 CNWT =
( CpWT WT)
WT

25 ng DNA
JAK2 V617F .
JAK2 V617F % =
CNV617F
(CNV617F + CNWT)
x 100
37

PC-VF JAK2 V617F
99,9%.
NC-VF JAK2 V617F
0,1%.
,
, 38, .

CN
JAK2 V617F JAK2
.
. .
, , .

.
, Frequently Asked
Questions ( )
: www.qiagen.com/FAQ/FAQList.aspx.
QIAGEN /-

(
, . , 47).

V617F
,

,
,
,
RQPCR, ,
PCR

.
ipsogen JAK2
MutaQuant 15 30C

. .
,
15.

.
38

,
PPM

.
PCR.
(H2O)
,
,
,
,

.
,

.

.

.
,
a)
F1/F2
530 nm/640 nm.
b)

.
PCR.
c)
.

Single ()

PCR.

DNA (OD260/OD280)
.

DNA.
39

a)

.
ipsogen JAK2
MutaQuant 15 30C

. .
,
15.

.
b)
DNA-
DNA
.
:

DNA,
.

.

.
,

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a)

.
LightCycler

F1/F2
530 nm/640 nm.
40

b)
,
,
PCR

,

,

.
c)

V617F
PPM
,
,

.

.
,

.

.

PPM
-

PPM DNA
41

LightCycler
480. ISO 13485:2003.

www.qiagen.com/support/.

.
,

, 12.

.

QIAGEN .

.
.
12 DNA

JAK2 V617F. 80
, 3 ipsogen
JAK2 MutaQuant.
Applied Biosystems 7500 Real-Time PCR.
15.
42
15. DNA
n*
(%)
(%)
95
73
0,004
117,5
0,000
0,015
0,05
80
0,101
89,2
0,003
0,284
0,5
79
0,449
61,6
0,161
0,950
68
1,169
41,6
0,611
1,998
80
2,046
33,5
1,168
3,185
80
3,733
30,6
2,120
5,560
77
5,246
22,4
3,647
7,309
12,5
80
16,633
16,6
12,792
22,335
31
80
28,602
14,8
22,705
34,773
50
76
56,181
6,6
50,024
63,724
78
80
80,153
3,8
75,352
85,551
100
70
99,998
0,003
99,992
100,000
JAK2 V617F
(%)
* .
3
3 (Box and
Whisker) .
n = , = .

(LOB)
(8 , 76 ). 0,014%.
(LOD)
, (7 , 68 ).
0,061%, 90% 0,091%.

10.000 JAK2 (
V617F).
43
JAK2 V617F 0,014% JAK2

V617F .
JAK2 V617F >0,014% <0,091%

( ).
JAK2 V617F 0,091%

JAK2 V617F .
12 ,
DNA
JAK2 V617F.
5 .
ipsogen JAK2 MutaQuant
.

DNA 87
ipsogen JAK2 MutaQuant. ,
JAK2 V617F
ipsogen
JAK2 MutaScreen EZ (. 673223).
16.
44
16.
ipsogen JAK2 MutaScreen EZ
ipsogen
JAK2 MutaScreen EZ

ipsogen JAK2
MutaQuant
40
40
49
21
21
17
17
45
87
100% (95% : 91%, 100%)
71% (95% : 51%, 85%)
89% (95% : 79%, 95%)
QIAGEN , online

QIAGEN.
-
, , .
, online
QIAGEN (Reference Database)
www.qiagen.com/RefDB/search.asp
QIAGEN .
45

1. National Center for Biotechnology Information (NCBI): NT_008413..
2. James, C. et al. (2005) A unique clonal JAK2 mutation leading to
constitutive signalling causes polycythaemia vera. Nature 434, 1144.
3. Levine, R. L. et al. (2005) Activating mutation in the tyrosine kinase JAK2 in
polycythemia vera, essential thrombocythemia, and myeloid metaplasia with
myelofibrosis. Cancer Cell 7, 387.
4. Kralovics, R. et al. (2005) A gainoffunction mutation of JAK2 in
myeloproliferative disorders. N. Engl. J. Med. 352, 1779.
5. Baxter, E. J. et al. (2005) Acquired mutation of the tyrosine kinase JAK2 in
human myeloproliferative disorders. Lancet 36, 1054.
6. Tefferi, A. et al. (2009) Myeloproliferative neoplasms: contemporary
diagnosis using histology and genetics. Nat. Rev. Clin. Oncol. 6, 627.
7. Prchal, J.F. and Axelrad, A.A. (1974) Bone marrow responses in
polycythemia vera. N. Engl. J. Med. 290, 1382.
8. Tefferi, A. and Vardiman, J.W. (2008) Classification and diagnosis of
myeloproliferative neoplasms: the 2008 World Health Organization criteria
and point-of-care diagnostic algorithms. Leukemia, 22, 14.
9. Barosi, G. et al. (2009) Response criteria for essential thrombocythemia and
polycythemia vera: result of a European LeukemiaNet consensus
conference. Blood 113, 4829.
10. Pardanani, A. et al. (2011) Safety and efficacy of TG101348, a selective
JAK2 inhibitor, in myelofibrosis. J. Clin. Oncol. 29, 789.
11. Lippert, E. et al. (2006) The JAK2-V617F mutation is frequently present at
diagnosis in patients with essential thrombocythemia and polycythemia vera.
Blood 108, 1865.
12. van der Velden, V.H. et al. (2003) Detection of minimal residual disease in
hematologic malignancies by real-time quantitative PCR: principles,
approaches, and laboratory aspects. Leukemia 17, 1013.
13. Branford, S. et al. (2006) Rationale for the recommendations for
harmonizing current methodology for detecting BCR-ABL transcripts in
patients with chronic myeloid leukaemia. Leukemia 20, 1925.

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In vitro o

,
www.qiagen.com/Support,
00800-22-44-6000
QIAGEN (.
www.qiagen.com).
47
. .
ipsogen JAK2
MutaQuant Kit (12)
12 :
- JAK2, JAK2 V617F,
PPM-WT,
PPM-VF
673522
ipsogen JAK2
MutaQuant Kit (24)
24 :
- JAK2, JAK2 V617F,
PPM-WT,
PPM-VF
673523
Rotor-Gene Q MDx IVD

PCR
Rotor-Gene Q MDx
5plex HRM Platform
PCR
Melt
5 (,
, , , )
HRM,
, ,
, 1
,

9002032
Rotor-Gene Q MDx
5plex HRM System
PCR
Melt
5 (,
, , , )
HRM,
, ,
, 1
,

9002033

,
QIAGEN. QIAGEN
www.qiagen.com.
QIAGEN .
48
49
50
51
52
in vitro . ipsogen ,
QIAGEN.
. QIAGEN
. .
QIAGEN , , ,
.
ipsogen . QIAGEN

.
JAK2 V617F ,
EP1692281, ... 7,429,456 7,781,199,
... US20090162849 US20120066776, .
JAK2V617F.
QIAGEN .
jak2licenses@qiagen.com.
: QIAGEN , HRM , ipsogen , MutaQuant , Pyrosequencing , Rotor-Gene ( QIAGEN). ABI PRISM , Applied
Biosystems, FAM, SYBR, TAMRA (Life Technologies). Excel (Microsoft Corporation). HybProbe, LightCycler, TaqMan ( Roche).

:
1.
ipsogen JAK2 MutaQuant ipsogen JAK2 MutaQuant

. QIAGEN
,
ipsogen JAK2 MutaQuant www.qiagen.com.
2.
, QIAGEN / /-
.
3.
,
.
4.
QIAGEN , .
5.

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/ .
, . www.qiagen.com.
2013 QIAGEN, .
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Sample & Assay Technologies

Ipsogen JAK2 MutaQuant Kit Handbook

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Ipsogen JAK2 MutaQuant Kit Handbook

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2013

QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden,

Sample & Assay Technologies

QIAGEN: Sample and Assay Technologies

qPCR RotorGene Q MDx 5plex HRM Rotor

qPCR ABI PRISM 7900HT SDS, Applied Biosystems 7500

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

1. (Hgb) >18,5 g.dl-1 () >16,5 g.dl-1

1. 450 x 109 l-1

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

ipsogen JAK2 MutaQuant 01/2013

V617F positive control (

V617F negative control (

M1-VF Standard Dilution (

M2-VF Standard Dilution (

M3-VF Standard Dilution (

M4-VF Standard Dilution (

WT-1 Standard Dilution (

WT-2 Standard Dilution (

ipsogen JAK2 MutaQuant 01/2013