AccuPower®

JAK2 V617F Quantitative PCR Kit

JAK-1111
 AccuPower® JAK2 V617F Quantitative PCR Kit

AccuPower® JAK2 V617F Quantitative PCR

Kit

Kit for detection of human JAK2 V617F mutation from

clinical samples through real-time quantitative PCR

User‟s Guide

Version No.: 2.0 (2011-03-10)

BIONEER CORPORATION
49-3 Munpyeong-Dong, Daedeok-Gu
Daejeon, 306-220, South Korea
Tel: +82-1588-9788
Fax: +82-42-930-8600
Email: order@bioneer.co.kr
www.bioneer.co.kr

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 AccuPower® JAK2 V617F Quantitative PCR Kit

Safety Warnings and Precautions

Please inquire BIONEER’s Customer Service Center to obtain a copy of
the Material Safety Data Sheet (MSDS) for this product.
Before, during and after use of this kit as described in this User
Manual, all potentially hazardous materials (i.e. materials that may have
come in contact with clinical samples) including tubes, tips and
materials should be processed and disposed of according to applicable
and appropriate regulations of the municipality/government in which
this product is being used.
Please read the User Manual before using this Kit. Please check the
integrity of all tubes, tips and other materials supplied with this kit prior
to use. Adhere to general clinical laboratory safety procedures during
the experiment.
The results yielded from this Kit may be used for preliminary analysis
only. This kit is not intended for human or veterinary diagnostics.
Some applications that may be performed with this kit may infringe
upon existing patents in certain countries. The purchase of this kit does
not include or provide a license to perform patented applications. Users
may be required to obtain a license depending on country and
application. We do not condone nor recommend the unlicensed use of
a patented application.

Warranty and Liability

All BIONEER products are manufactured and tested under strict
quality control protocols certified under ISO 9001 and ISO 13485
standards. BIONEER guarantees the quality of all directly manufactured
products during the warranty period of one (1) year from date of
purchase. If any issues are discovered relating to compromise in
product quality, immediately contact BIONEER’s Customer Service
Center (order@bioneer.com).

Registered Trademarks
AccuPower® is a registered trademark of Bioneer Corporation, Republic of Korea.
Copyright © 2011 by Bioneer Corporation. All rights reserved

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 AccuPower® JAK2 V617F Quantitative PCR Kit

TABLE OF CONTENTS
1. INTENDED USE………………………………………………..………….5
2. INTRODUCTION……………………………….…………………..…….6
3. FEATURES AND PRINCIPLE OF THE TEST……………………….6
3.1 Kit Format………….………...………………………….........……….8
3.2 Sensitivity and Dynamic Range..………….……………………8
3.3 Endogenous Internal Control (EIC)……….……………………8
4. CONTENTS OF THE KIT……………….….……………..…………..10
5. STORAGE………………………….………….…………………………..11
6. REQUIRED MATERIALS AND EQUIPMENT (NOT PROVIDED
IN THE KIT)..…………………………...……………………………......11
7. GENERAL PRECAUTIONS.……………...……………………………12
8. PROTOCOL….…………….………………….…………………………..13
8.1. Preparation: Specimen collection, storage, and
transport..……………………………………….…………………...13
8.1.1. Specimen collection.………………..….…………………….13
8.1.2. Sample storage..…..………………..……..…………………..13
8.1.3. Sample transport………………..…………………………….14
8.1.4. Interfering Substances…………...………………………….14
8.2. Work Flow Schema…..…………………………………………..15
8.3. DNA Extraction…..………………….……………………………...16
8.4. Experiment Preparation………..………………………………..16

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 AccuPower® JAK2 V617F Quantitative PCR Kit

8.5. Programming the Exicycler™ 96 Real-Time Quantitative

Thermal Block.………..……………………………………….……18
9. DATA ANALYSIS….…….…………………………………………….. 23
9.1 Process of data analysis……………………………………….…23
9.2 Analysis Result …………….………………………………………..27
10. TROUBLESHOOTING…...………………………………………...…. 29
11. REFERENCES …………………………………………….…………….. 31
12. SYMBOLS …………………………………………………………...….. 32

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 AccuPower® JAK2 V617F Quantitative PCR Kit

1. INTENDED USE
AccuPower® JAK2 V617F Quantitative PCR kit is designed for
detection of human JAK2 V617F mutation from clinical samples,
through real-time quantitative PCR (RT-qPCR).

This kit is optimized for use with BIONEER’s Exicycler™ 96

Real-Time Quantitative Thermal Block (Cat. No: A-2060).

This Kit is designated for research use only (RUO), and thus is
not intended for use in diagnostic procedures. Results yielded
by this Kit may only be used for preliminary analysis. The use of
kit is only for qualified users trained to correctly and safely
handle clinical specimens and conduct molecular biological
experiments. All waste generated before, during and after the
experiment should be processed in compliance with local
regulations.

Please read the instructions contained within this manual

thoroughly before using this Kit and check the integrity of all
components before use.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

2. INTRODUCTION

The JAK2 genetic mutation is observed at high frequency in

MPD patients. After recent studies have revealed the relationship
between MPD and the JAK2 point mutation, many assays have
been developed to screen for the JAK2 mutation (JAK2 V617F)
for diagnosis of MPD.
The JAK2 V617F mutation can be found in 65~97% of
polycythemia vera cases, 23~57% in thrombocythemia cases,
and 43~50% in idiopathic myelofibrosis cases.
The JAK2 V617F mutation can be screened through
conventional PCR or sequencing. The drawbacks of these
methods include difficulties in keeping blood samples or
decreased specificity. The qPCR method of detection delivers
high sensitivity and fast results due to the absence of a post-
PCR process.
BIONEER’s AccuPower® JAK2 V617F Quantitative PCR Kit
can deliver sensitive and reproducible JAK2 V617F screening
results that are difficult to obtain with other detection methods.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

3. FEATURES AND PRINCIPLE OF THE TEST

Real-time PCR involves the selective amplification of a target
DNA sequence while monitoring the progress of amplification in
real-time through a visualizing agent. The specificity is provided
by a pair of sufficient length primers, along with a hydrolysis
probe which is also sequence specific. Monitoring amplified
product is conducted by labeling the hydrolysis probe with a
matched pair of fluorescent dyes (5’-FAM and 3’-BHQ1), which
through fluorescence resonance energy transfer (FRET) and the
inherent 5’ – 3’ exonuclease activity of the DNA polymerase, will
emit a specific wavelength of light within the visible spectrum
(520 nm) when cleaved after binding to the amplicon.

The Kit was designed to maximize reproducibility and ease-of-

use by lyophilizing the primers, probes, DNA polymerase, dNTPs
and salts using our proprietary stabilization technology to
preserve the full activity of the mixed reagents. The primer-
probe set was selected from a pool of primer-probe
combinations designed by our sophisticated bioinformatics
algorithms, to 1) achieve maximum amplification efficiency, and
2) as with all of our other AccuPower® Diagnostic Kits, match
the thermal cycle so that this Kit could be run simultaneously
with other Kits from our AccuPower® Diagnostic Kit series.

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AccuPower® JAK2 V617F Quantitative PCR Kit

3.1. Kit Format

AccuPower® JAK2 V617F Quantitative PCR kit is a ready-to-
use product that can analyze up to 48 samples in a single run.

3.2. Sensitivity and Dynamic Range

The sensitivity and the dynamic range of AccuPower® JAK2
V617F Quantitative PCR kit are 102 copies/ reaction and 8 logs,
respectively.

3.3 Endogenous Internal Control (EIC)

AccuPower® JAK2 V617F Quantitative PCR kit employs
Endogenous Internal Control (EIC; GAPDH) amplification in all
wells to confirm correct PCR amplification.

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AccuPower® JAK2 V617F Quantitative PCR Kit

Principle of a Real-Time PCR employing a 5‟-nuclease probe

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 AccuPower® JAK2 V617F Quantitative PCR Kit

4. CONTENTS OF THE KIT

Cat. No.
Labeling & Content
JAK-1111

① JAK2 Wild/Mutant Premix

Each 48 tubes
8 well-strip in aluminum foil bag

② JAK2 Mutant standard DNA (102~105 copy), 15 ul / tube

PC, NC x 24 tubes

③ JAK2 Wild standard DNA (102~105 copy), 15 ul / tube

PC, NC x 24 tubes
15 ul / tube
④ PCR grade water (for NTC)
x 24 tubes
1800 ul x 4
⑤ PCR grade water
tubes
Optical Sealing film 1 sheet
Quick Manual 1 sheet

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 AccuPower® JAK2 V617F Quantitative PCR Kit

5. STORAGE
AccuPower® JAK2 V617F Quantitative PCR kit should be
stored at ≤- 20℃ away from UV/sunlight. The Kit is guaranteed
stable until the expiration date printed on the label. Repeated
freeze/thaw cycles (more than twice) should be avoided, as it
may affect Kit quality. If intermittent use of the Kit is expected,
components should be frozen in aliquots. Reagents should not
be stored at 4℃ for more than 2 hours.

6. REQUIRED MATERIALS AND EQUIPMENT

(NOT PROVIDED IN THE KIT)

- Disposable powder-free gloves, medical grade

- Genomic DNA extraction kit or equivalent protocol and
materials necessary to extract DNA (see 8.3 DNA
Extraction)
- Appropriate capacity pipettes or multi-channel pipettes
- Sterilized pipette tips (filtered)
- Vortex and microcentrifuge capable of handling 8-well
strip-tubes (e.g. ExiSpin™ Programmable Vortex/
Microcentrifuge (Cat. No: A-7040))
- Exicycler™ 96 Real-Time Quantitative Thermal Block (Cat.
No: A-2060)

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 AccuPower® JAK2 V617F Quantitative PCR Kit

7. GENERAL PRECAUTIONS

- Store all kit components at -20 °C

- Always wear gloves and a mask when handling
biohazardous agents
- DO NOT repeatedly freeze/thaw Kit components

- Always use sterile, filtered pipette tips

- Clinical samples, their derivatives and positive controls
should be stored in a separate location/freezer from where
the rest of the Kit components are stored
- All positive controls should be added in a physically
separate location from where the premix is reconstituted

- All Kit components should be allowed to slowly thaw for at

least 30 minutes before initiating an experiment

- Briefly vortex and spin-down all Kit components after

thawing to ensure optimum results

- DO NOT reuse opened reagents, nor mix reagents from
different production lots

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 AccuPower® JAK2 V617F Quantitative PCR Kit

8. PROTOCOL

8.1. Preparation: Specimen collection, storage, and transport

Caution: All samples must be treated as potential biohazards.

Attention: We recommend genomic DNA extracted from

human whole blood for the best results. We strongly
recommend that the genomic DNA used for the
assay be of the following quality:
Concentration: 30 ng/ul; Purity (A260/280): >1.7

8.1.1. Specimen collection

AccuPower® JAK2 V617F Quantitative PCR kit is optimized
for genomic DNA extracted from human whole blood
samples. For blood withdrawal, standard specimen collection
tubes such as BD SST Serum Separator tubes or disposable
tubes containing EDTA as anticoagulant can be used.

8.1.2. Sample storage

Do not store whole blood at 2~25℃ for over 1 day. For
long term storage of clinical samples, please aliquot and
store at -70℃.

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AccuPower® JAK2 V617F Quantitative PCR Kit

8.1.3. Sample transport

All samples should be transported in a shatterproof
transport container to prevent potential infection from
sample leakage. Samples should be transported according to
local/national guidelines regarding biohazard transportation.

8.1.4. Interfering substances

Heparin (≥ 10 IU/㎖) is a known inhibitor of PCR. Samples
that have been collected in tubes containing heparin should
not be used. In addition, samples from heparin-treated
patients should not be used. Clinical samples may contain a
variety of PCR inhibitors. For efficient PCR, such inhibitors
must be removed during the DNA extraction and purification
process.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

8.2. Work Flow Schema

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 AccuPower® JAK2 V617F Quantitative PCR Kit

8.3. DNA Extraction

Genomic DNA must be extracted from raw clinical samples
before this real-time PCR experiment can be conducted. Please
perform genomic DNA extraction according to pre-established
protocols, or, in the case of kit extraction of genomic DNA,
adhere to the manufacturer protocol for DNA extraction. For
the best results, we recommend the following kits:

Product Cat. No. Platform

AccuPrep® Genomic DNA
K-3032 Manual
Extraction Kit
ExiPrepTM Blood Genomic DNA
K-3215 ExiPrepTM 16 Plus
Kit

8.4. Experiment Preparation

① Considering the quantity of samples and controls, take out
the appropriate number of wild and mutant premix strips,
and also take out 1 strip of positive control, negative
control and standard tubes each.

② Each well requires 45㎕ PCR Grade water. Aliquot 45㎕ in

each well.

③ Pipette 5 ul of extracted DNA in both wild and mutant

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AccuPower® JAK2 V617F Quantitative PCR Kit

tubes, and place the controls in the tubes listed below.

After pipetting everything, seal the wells with the optical
sealing film using the applicator. (In order to prevent
contamination, add the sample DNA in a separate location)
Control Name Wild Mutant
Non-Template Control (NTC) A1 A7
Positive control (PC) B1 B7
Negative Control (NC) C1 C7
Standard D1~H1 D7~H7

④ To mix the tube thoroughly (to dissolve the Premix pellet

and mix the DNA), use the ExiSpinTM (Cat.No : A-7040,
Bioneer Co., KOREA) and perform 20 cycles at 2500 rpm
for 5 sec. spindown / 20 sec. hard vortex.

⑤ While the ExiSpinTM is operating, start the ExicyclerTM 96

program and configure the settings.

⑥ After the ExiSpin™ cycling is complete, we recommend

placing the tubes in the Exicycler™ 96 immediately.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

8.5. Programming the Exicycler™ 96 Real-Time Quantitative

Thermal Block
① Turn the Standby Power Switch, located at the rear of the
instrument ON. The front ring-LED status light should turn
on RED (Fig.1-①).

Fig. 1) Front view of the instrument showing operating buttons

② Press the front Operation Power Switch for 2 seconds. A

brief self test sequence will initiate. If the self test passes,
the front ring-LED will blink GREEN (Fig.1-②), with a short
beep.

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AccuPower® JAK2 V617F Quantitative PCR Kit

③ Push the Door Switch for 2 seconds to slide the 96-well

thermal block out. Insert the reaction tubes. After sample
loading is complete, push the Door Switch for 2 seconds
to close the door.

Fig. 2) Side view and the 96-well thermal block of the instrument

④ Use a spreadsheet program capable of creating ‘*.xls‟

files and create a sample layout map (shown below).
Wells A1 to H1 are pre-configured for controls. Do not fill
out well information for those wells. For JAK2, only lanes
1~6 should be filled out.

Fig. 3) Sample Layout Map

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AccuPower® JAK2 V617F Quantitative PCR Kit

⑤ Save the sample layout map file as an ‘*.xls’ file. Save the
file in an easily findable location.

⑥ Double click the ‘ExiGenotyper Run’ icon to start the

program. If a ‘Device is Not ready’ warning appears, please
close and restart the program. If a ‘System is ready’
prompt appears, the computer has successfully connected
to the instrument.

⑦ To setup the protocol for PCR, click „File - Design

Experiment‟ from the top menu bar.

⑧ When the „Select Diagnostics Kit‟ window appears,

select ‘JAK2 V617F Quantitative Rea-Time PCR kit’.

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AccuPower® JAK2 V617F Quantitative PCR Kit

⑨ After selecting the kit, find the sample layout map saved
in step 5 and click the „Open‟ button.

⑩ When the ‘Sample Assign View’ window appears, verify

that the physical locations of the strips within the
instrument match the layout map. If the layout and
locations match, click the „OK‟ button.

⑪ When the ‘Quick Start…’ window appears, verify sample

layout and PCR protocol information. Click „OK‟ if
everything is correct.

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AccuPower® JAK2 V617F Quantitative PCR Kit

⑫ To perform the reaction, click the ‘▶‘button located

within the top toolbar.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

9. DATA ANALYSIS
9.1. Process of data analysis
① Data can be analyzed by opening the data file through the
‘ExiGenotyper Analysis’ program, which icon is located on
the computer Desktop screen. For details on how to analyze
data using the data analysis program, refer to the instrument
Instruction Manual.
② Select File>Open and select the data file (.ex3) to analyze.
The data files save in „C:\ExiGenoData\GUEST‟ folder.

③ Several tabs are displayed as shown below. The Final Result

tab is configured to be initially displayed.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

④ The Final Result tab displays results for both wild type and
mutant measurements in spreadsheet format. The Counted
Mutant ratio (%) is also displayed. Results for JAK2 Wild and
JAK2 Mutant are contained within their corresponding tabs.

*JAK2 V617F mutation ratio calculation method

JAK2 V617F % = [JAK2 V617F / (JAK2 V617F + JAK2 WT)] * 100

⑤ The experiment fluorescence graph can be viewed in the Flu.

Graph tab.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

⑥ The Well vs Ct tab has the distribution of samples by Ct

value.

⑦ The Standard curve tab displays standard curve.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

⑧ The Plate Info tab contains information about all sample

names (including NTCs, PCs and NCs) and their assignments.

⑨ The Flu. Graph, Well vs Ct, Plate Info tab images can be
saved as *.jpg files. Use the save image icon ( ) within the
toolbar to save the image.

⑩ The Result tab contents can be exported as a spreadsheet

(*.xls). Select the save spreadsheet icon ( ) within the
toolbar to save the spreadsheet.

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AccuPower® JAK2 V617F Quantitative PCR Kit

9.2. Analysis Results

Wild “G” (copies) Mut. “T” (copies) Ratio (%)

Sample 1 27585.3 554758.56 95.26
Sample 2 41770.52 521600.14 92.59
Sample 3 88480.53 1654454.44 94.92
Sample 4 186254.28 430030.6 69.78
Sample 5 142199.75 300376.67 67.87
Fig 4) Detection of FAM signal (JAK2 V617F Wild/Mutant) and
Mutation ratio of samples
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AccuPower® JAK2 V617F Quantitative PCR Kit

Table 1. Cut-off table for data analysis

FAM
Type
NTC (JAK2 V617F- Result
Wild/Mutant)

Cut-off value - < 35 Ct Valid

Positive/Negative
- >90% Valid
ratio
Positive/Negative
- <90% Repeat
ratio
Negative/positive
- >90% Valid
Control ratio
Positive/Negative
- <90% Repeat
ratio
Sample + -/+,+/- Repeat

Sample - -/+, +/-, +/+ Valid

Sample - -/- Repeat

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 AccuPower® JAK2 V617F Quantitative PCR Kit

TROUBLESHOOTING
Comments and suggestions
There is no FAM fluorescence signal in both the unknowns and
standards.
• Thermal cycle program error
☞ Confirm that the correct thermal cycle was
programmed.
See 8.5. Programming the Exicycler™ 96 Real-
Time Quantitative Thermal Block.
If the • Omission of reaction component(s)
TAMRA (IPC)
☞ Review your reaction preparation procedure.
fluorescence
See 8.4. Experiment Preparation.
signal was
detected • incorrect appointment of reporter and quencher
dyes
☞ Check the appointments of the reporter and
quencher dyes.
See 8.5. Programming the Exicycler™ 96 Real-
Time Quantitative Thermal Block.
• An error may have occurred during genomic DNA
extraction.

If the ☞ Review your genomic DNA extraction procedure.

TAMRA (IPC) • The kit may have spoiled, due to bad storage or
fluorescence expiration.
signal is ☞ Assess your storage conditions and review the
undetectable expiration date.
Repeat the assay with new reagents in duplicate.
See 5. Storage.

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AccuPower® JAK2 V617F Quantitative PCR Kit

A weak signal is present in the unknowns and standards

• The reaction mixture has been incorrectly concocted.
☞ Review your reaction preparation procedure.
See 8.4. Experiment Preparation.

• Inhibition of PCR
☞ Review your genomic DNA extraction procedure.

• Reagent degradation from repeated freeze/thaw cycles.

☞ Assess your storage conditions and remaining kit components.

• The Kit has passed its expiration date.

☞ Review the expiration date printed on the Kit.

A signal is present in the NTC.

• Contamination may have occurred
☞ Review your workspace and assess for contamination.

Signals are inconsistent between samples.

• There may have been a pipetting error
☞ Review the pipetting technique and calibration.

• Cross contamination may have occurred

• There may have been contamination on the optical adhesive cover

☞ Review your workspace and assess for contamination.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

11. REFERENCES

Widespread occurrence of the JAK2 V617F mutation in chronic

myeloproliferative disorders Blood, 2005 106: 2162-2168

Acquired mutation of the tyrosine kinase JAK2 in human

myeloproliferative disorders. Lancet , 2005; 365: 1054–61

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic

Erythrocytosis N Engl J Med, 2007;356:459-68.

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 AccuPower® JAK2 V617F Quantitative PCR Kit

12. SYMBOLS

Catalog Number

Batch code

Contains Sufficient for test

Expiration Date

Storage Conditions (Temp.)

Manufacturer

Caution, Consult accompanying documents

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AccuPower® JAK2 V617F Quantitative PCR Kit

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