PNAClamp™ EGFR Mutation Detection Kit (Ver.2) · Instruction manual for product # PNAC-3002 ... ABI ABI 7500 ABI ABI 7900 ABI StepOnePlus Qiagen Rotor-Gene Q ABI QuantStudio 5

PNAClamp™ EGFR Mutation Detection Kit (Ver.2)

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For in vitro diagnostic use

Instruction manual for product # PNAC-3002

Version 2.0

Store at -15℃ to -20℃

Instruction Version: 2.0

Date of Revision: Sep. 2016


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Contents

Product Name ................................................................................................................ 3

Intended Use .................................................................................................................. 3

Principle and Overview ................................................................................................ 5

Contact Information ..................................................................................................... 6

Safety Information ........................................................................................................ 6

Equipment and materials supplied by the user .......................................................... 6

Warnings and Precuations ........................................................................................... 7

Storage Condition and Stability ................................................................................... 8

Kit contents .................................................................................................................... 8

Procedures ...................................................................................................................... 9

1. DNA preparation ........................................................................................................................ 9

2. Preparation of the Real-Time PCR Mixture ......................................................................... 10

3. Real-Time PCR reaction ......................................................................................................... 11

4. Assessment ................................................................................................................................ 11

Examples of Analysis ................................................................................................... 14

Quality Control ............................................................................................................ 23

Performance Test ......................................................................................................... 23

References .................................................................................................................... 24

Explanation of Symbols on the Label ........................................................................ 25

Endnotes ....................................................................................................................... 26


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PRODUCT NAME

Product Name: PNAClamp™ Mutation Detection Kit

Brand Name: PNAClamp™ EGFR Mutation Detection Kit (Ver.2)

INTENDED USE

The PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is to detect 40 somatic mutations in the epidermal

growth factor receptor (EGFR) gene (Table 1).

The kit is to be used by trained laboratory professionals within a laboratory environment using, for example,

DNA extracted from formalin-fixed paraffin-embedded samples of lung biopsies and surgical tissue samples.

The kit is for in vitro diagnostic use.

Please read the instructions carefully prior to use.

The PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is a CE marked diagnostic device in accordance with

the European Union in vitro Diagnostic Medical Device Directive 98/79/EC.

It is MFDS approved for clinical use in Korea.


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Table 1. EGFR mutations detected by this kit

No. Reagent Exon Amino Acid Change Nucleotide change Cosmic No.

1 G719X PNA mix

18 p.G719A c.2156G>C 6239

18 p.G719S c.2155G>A 6252

18 p.G719C c.2155G>T 6253

2 E19 del. PNA mix

19 p.E746_A750del c.2235_2249 del 15 6223

19 p.E746_T751>I c.2235_2252>AAT 13551

19 p.E746_T751del c.2236_2253 del 18 12728

19 p.E746_T751>A c.2237_2251 del 15 12678

19 p.E746_S752>A c.2237_2254 del 18 12367

19 p.E746_S752>V c.2237_2255>T 12384

19 p.E746_A750del c.2236_2250 del 15 6225

19 p.E746_S752>D c.2238_2255 del 18 6220

19 p.L747_A750>P c.2238_2248>GC 12422

19 p.L747_T751>Q c.2238_2252>GCA 12419

19 p.L747_E749del c.2239_2247 del 9 6218

19 p.L747_T751del c.2239_2253 del 15 6254

19 p.L747_S752del c.2239_2256 del 18 6255

19 p.L747_ A750>P c.2239_2248

TTAAGAGAAG>C 12382

19 p.L747_P753>Q c.2239_2258>CA 12387

19 p.L747_T751>S c.2240_2251 del 12 6210

19 p.L747_P753>S c.2240_2257 del 18 12370

19 p.L747_T751del c.2240_2254 del 15 12369

19 p.L747_T751>P c.2239_2251>C 12383

19 p.E746_A750>IP c.2235_2248>AATTC 13550

19 p.E746_T751>V c.2237_2252>T 12386

19 p.E746_T751>IP c.2235_2251>AATTC 13552

19 p.E746_S752>I c.2235_2255>AAT 12385

19 p.E746_P753>VS c.2237_2257>TCT 18427

19 p.L747_T751del c.2238_2252 del 15 23571

19 p.L747_S752>Q c.2239_2256>CAA 12403

3 T790M PNA mix 20 p.T790M c.2369C>T 6240

4 S768I(V2) PNA mix 20 p.S768I c.2303G>T 6241

5 E20 Ins.3dup PNA

mix

20 p.H773_V774insH c.2319_2320 insCAC 12377

20 p.P772_H773insTTP c.2315_2316

insGACAACCCC

20 p.P772_H773insGNP c.2315_2316

insGGGCAACCC

20 p.H773L c.2318A>T 13005

20 p.H773_V774insPH c.2319_2320 insCCCCAC 12380

20 p.V774_C775insHV c.2321_2322 insCCACGT 18432

6 L858R PNA mix 21 p.L858R c.2573T>G 6224

21 p.L858R c.2573_2574TG>GT 12429

7 L861Q PNA mix 21 p.L861Q c.2582T>A 6213

* del: deletion mutation, ins: insertion mutation

Cosmic Numbers are taken from ‘The Catalogue of Somatic Mutations in Cancer’.

(http://cancer.sanger.ac.uk/cosmic)


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PRINCIPLE AND OVERVIEW

The PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is based on peptide nucleic acid (PNA)-mediated real-

time PCR clamping technology. PNA is a synthetic DNA analog in which the phosphodiester backbone is

replaced by a peptide-like repeat formed by (2-aminoethyl)-glycine units.

PNA-mediated real-time PCR clamping relies on the following two unique properties of PNA probes. First,

PNA will hybridize to its complementary DNA target sequence only if the sequence is in complete match. Since

PNA/DNA duplexes are more thermodynamically stable than the corresponding DNA-DNA duplexes, even

with a single mismatch, PNA will not bind to complementary DNA strand, unlike DNA. Second, PNA oligomers

are not recognized by DNA polymerases and will not be utilized as primers in subsequence real-time PCR.

Instead, it serves as a sequence-selective clamp that prevents amplification during subsequent PCR.

When there is a mutation in target gene and therefore a mismatch is present, the DNA/PNA duplex is

destabilized, allowing strand elongation from a bound DNA oligomer which serves as a PCR primer. The

outcome is the positive reaction in real-time PCR from the samples harboring mutant allele, while amplification

of the wild-type gene is suppressed.

Figure 1. Principle of the PNAClamp™ EGFR Mutation Detection Kit (Ver.2)

The kit can rapidly detect EGFR mutation (within 2 h) with high sensitivity even with a small amount of DNA

(25~50 ng). The detection limit of the kit, when the mutated gene is mixed with wild-type DNA, is less than

1%.


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CONTACT INFORMATION

For any questions including technical support or concerns, please contact the distributors or the manufacturer.

Manufacturer:

EC Representative:

SAFETY INFORMATION

Material Safety Data Sheet (MSDS) is available upon request.

EQUIPMENT AND MATERIALS SUPPLIED BY THE USER

Reagents and equipment for DNA extraction

0.2 ml DNase-free PCR tubes or plates

Pipettes

A real-time PCR instrument fitted with a detector enabling evaluation of SYBR Green dye

Table 2. List of compatible real time PCR instruments

Company Model

Bio-Rad CFX 96

Roche Light cycler 480 II

ABI ABI 7500

ABI ABI 7900

ABI StepOnePlus

Qiagen Rotor-Gene Q

ABI QuantStudio 5

For other instruments, minor optimization might be necessary.

PANAGENE Inc.

54, Techno 10-ro, Yuseong-gu, Daejeon, 34027, Korea

E-mail: [email protected]

Tel: +82-42-861-9296

MT Promedt Consulting GmbH

Altenhofstrasse 80, 66386 St. Ingbert, Germany

E-mail: [email protected]

Tel: +49 (0) 6894 581020

mailto:[email protected]

mailto:[email protected]


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WARNINGS AND PRECUATIONS

Please read the instruction carefully and become familiar with all components of the kit prior to

use.

PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is for in vitro diagnostic use.

All experiments should be performed under proper sterile conditions with aseptic techniques. It

recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and

during the preparation of reagents.

Always wear powder-free gloves when you handle the kit.

To avoid repeated freezing and thawing, aliquot all reagents into appropriate volumes and store frozen

until use. Thaw appropriate volumes of reagents before each experiment.

All experimental procedures should be performed at room temperature. However, exposing EGFR PNA

2X premix at room temperature should be minimized for the optimal amplification.

Dissolve reagents completely and mix them thoroughly by vortex.

The EGFR PNA 2X premix solution contains fluorescence dye and should be kept dark.

If DNA has been extracted from a paraffin block, additional purification steps may be required.

PCR tubes should be weakly centrifuged before use.

Using non-recommended volume for reagent not only result in loss of performance but also increase the

chance of false result.

Using non-recommended volume and concentration for target DNA sample not only result in loss of

performance but also increase the change of false result.

Do not exchange and mix up different lots or other manufacture’s product.

Upon using instruments, use only recommended consumables only. If not, instruments will not be usable

or false result may prominent.

Additional validation testing by user may necessary when using non-recommended instruments.

Do not re-use any remaining reagents after PCR amplification is completed.

Do not use the reagents beyond the expiry date.


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STORAGE CONDITION AND STABILITY

The PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is shipped on ice package and must still be frozen on

arrival. If the kit is not frozen on arrival please contacts PANAGENE Inc. or the local distributor.

The PNAClamp™ EGFR Mutation Detection Kit (Ver.2) should be stored immediately upon receipt at -15℃

to -20℃. When stored under the recommended storage conditions in the package, the kit is stable until the

labeled expiration date.

After opening the kit, shelf-life is 3 months.

KIT CONTENTS

Store at -15℃ to -20℃

No. Name of component Description Volume Cap label

1 Non PNA mix Primers only 100 µl EGFR

1

2 G719X PNA mix G719X PNA and primers 100 µl EGFR

2

3 E19 del. PNA mix E19 del. PNA and primers 100 µl EGFR

3

4 T790M PNA mix T790M PNA and primers 100 µl EGFR

4

5 S768I(V2) PNA mix S768I PNA and primers 100 µl EGFR

5

6 E20 Ins. 3 dup PNA mix E20 Ins. 3 dup PNA and primers 100 µl EGFR

6

7 L858R PNA mix L858R PNA and primers 100 µl EGFR

7

8 L861Q PNA mix L861Q PNA and primers 100 µl EGFR

8

9 EGFR PNA 2X premix PCR reaction premix 1,250 µl /vial,

2 vials EGFR

2X premix

10 Clamping control Wild-type DNA 600 µl EGFR

Control

* Each kit contains enough material to test 25 DNA samples for all mutations.


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PROCEDURES

Figure 2. Workflow of the PNAClamp™ EGFR Mutation Detection Kit (Ver.2)

1. DNA preparation

Specimen collection and DNA extraction reagents are not included in the kit so they should be provided by

the user.

1) Paraffin embedded tissues or biopsy tissues can be used as specimens.

2) Specimen transport: Use standard pathology methodology to ensure specimen quality.

3) For DNA extraction Kit is recommended below.

Model Company Catalog number

High Pure PCR Template Preparation Kit Roche Diagnostics 11796828001

QIAamp DNA FFPE Tissue Kit Qiagen 56404

QIAamp DNA Mini Kit Qiagen 51304

Maxwell® 16 FFPE Plus LEV DNA Purification Kit Promega AS1135

4) Extracted DNA can be stored at 4℃ for up to 24 hours, or at -20℃ for long term storage.


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2. Preparation of the Real-Time PCR Mixture

Table 3. Set up reaction mixture per on reaction.

Components Volume

EGFR PNA 2X Premix (#9) 10 µl

Each PNA mix (#1~#8) 3 µl

Extracted DNA (25~50 ng total) or Clamping control (#10) 7 µl

Total volume 20 µl

1) Prepare 8 PCR tubes for one set of DNA samples to be tested. Label them as S1, S2, S3, S4, S5, S6,

S7 and S8. Prepare another set of 7 tubes for Clamping control (wild-type DNA) and label them as

C1~C8.

2) Add 10 µl of EGFR PNA 2X Premix (#9 from the kit) to each tube.

3) For each PCR tube, add 3 µl of corresponding PNA mix from #1~8 from the kit. For example, S1 and

C1 tubes will have #1 Non PNA mix, S2 and C2 tubes will have #2 G719X PNA mix and so forth.

4) For S1~S8 PCR tubes, add 7 µl of prepared DNA sample (25~50 ng total) to each tube to yield 20 µl

final volume.

5) For C1~C8 PCR tubes, add 7 µl of Clamping control (#10 from the kit).

6) If you have more than one DNA sample to be tested, prepare one set of Clamping control for the entire

experiment. In such case, it is recommended to prepare a master mix containing 2X Premix and each

PNA mix for all the samples and to aliquot 13 µl to each PCR tube.

7) When all reagents are loaded, tightly close/seal the PCR tube or 96 well plate. Otherwise, any

remaining reagents may evaporate.



3. Real-Time PCR reaction

Perform real-time PCR using the cycling conditions described below

ONE CYCLE

Pre-denaturation 94℃ 5 min

FOUR-STEP CYCLING (40 CYCLES)**

Denaturation 94℃ 30 sec

PNA clamping 70℃ 20 sec

Annealing 63℃ 30 sec

Extension* 72℃ 30 sec

* Set up the detection for reading SYBR Green at 72℃.

** If you use Light Cycler 480 II, Please set up 45 cycles for four-step cycling.

4. Assessment

* Refer to the specialized instrument user guide by Panagene for detail analysis method.

1) Clamping control (wild-type DNA control)

(1) Determine Ct value from each PCR reaction. The cycle number at which a signal is detected above

background fluorescence is termed as the cycle threshold (Ct).

(2) The Ct values of the Clamping control (tube C1~C8) should fall in the range given in Table 4.

The assay should be repeated if the values are not in recommended range.

Table 4. The acceptable Ct ranges of Clamping control

Assay Acceptable Ct range

① Non PNA mix (C1) ≤ 30

Assay Acceptable ΔCt-1* range

② G719X PNA mix (C2) < 2

③ E19 del. PNA mix (C3) < 2

④ T790M PNA mix (C4) < 2

⑤ S768I(V2) PNA mix (C5) < 2

⑥ E20 Ins. 3 dup PNA mix (C6) < 2

⑦ L858R PNA mix (C7) < 2

⑧ L861Q PNA mix (C8) < 2

*ΔCt-1 = [Standard Ct] – [Sample Ct or Clamping control Ct], Standard Ct values given in Table

6 below.



2) DNA samples

(1) Determine Ct values of each sample (S1~S8).

i. Ct value of Non PNA mix (S1) should be 22~35.

ii. Ct value of Non PNA mix (S1) can serve as an internal control to indicate the purity and the

concentration of DNA. Thus, the validity of the test can be decided by the Ct value of Non

PNA mix (S1) as shown in Table 5.

Table 5. The acceptability of samples

Acceptability Ct value of Non

PNA mix(S1) Descriptions and recommendations

Optimal 22< Ct <30 The amplification and the amount of DNA sample are

optimal.

Acceptable 30≤ Ct <35

The target gene was amplified with low efficiency. For

more reliable result, it is suggested that repeat PCR

reaction with a higher amount of DNA.

Invalid

Ct ≤22 Possibility of false positive is high. Repeat the PCR

reaction with a lower amount of DNA.

35≤ Ct The amplification was failed. Check DNA amount and

purity. New DNA prep might be required.

(2) Calculate the ΔCt-1 values by subtracting the sample Ct values (or Clamping control Ct value)

from the Standard Ct values given in Table 6. If the Ct of samples is displayed as NA (not

applicable), then set Ct value as 38 for further calculation.

*ΔCt-1 = [Standard Ct] – [Sample Ct (S2, S3, S4, S5, S6, S7 or S8) or Clamping control Ct]

Table 6. The value of Standard Ct

Instruments

Standard Ct

G719X

PNA mix

E19 del.

PNA mix

T790M

PNA mix

S768I(V2)

PNA mix

E20 Ins. 3

dup PNA

mix

L858R

PNA mix

L861Q

PNA mix

Bio-Rad CFX96 33.5 34 33 33 30 33 33

Roche LC480 33.5 34.5 33 33 30 33 33

ABI 7900 33.5 34.5 33 33 30 33 33

ABI 7500 33.5 34 33 33 30 33 33

ABI StepOnePlus 33.5 34.5 33 33 30 33 33

Rotor-Gene Q 33.5 34 33 33 30 33 33

QuantStudio 5 33.5 34.5 34 33 30 33.5 33

(3) Calculate ΔCt-2 [Ct value of sample subtracted by Ct value of Non PNA mix].

**ΔCt-2 = [Sample Ct (S2, S3, S4, S5, S6, S7 or S8)] – [Non PNA mix Ct (S1)]



(4) Assess the result along with the values of ΔCt-1 and ΔCt-2 as given in Table 7.

Table 7. Assessment of the result

ΔCt-1 ΔCt-2 Assessment

2≤ ΔCt-1 ΔCt-2 ≤8 Mutant

8< ΔCt-2 Wild

0< ΔCt-1 <2 ΔCt-2 ≤3 Mutant

3< ΔCt-2 Wild

ΔCt-1 ≤0 All value Wild



EXAMPLES OF ANALYSIS

1. Using Bio-Rad CFX96

1) Profile of Clamping control

Assay Clamping control Ct Accep. range Result

① Non PNA mix (C1) 22.47 ≤ 30 Acceptable

Assay Clamping

control Ct ΔCt-1

Accep.

ΔCt-1 range Result

② G719X (C2) 35.48 -1.98 < 2 Acceptable

③ E19 del. (C3) 35.03 -1.03 < 2 Acceptable

④ T790M (C4) 34.66 -1.66 < 2 Acceptable

⑤ S768I(V2) (C5) 32.86 0.14 < 2 Acceptable

⑥ E20 Ins. 3 dup (C6) 31.88 -1.88 < 2 Acceptable

⑦ L858R (C7) 34.21 -1.21 < 2 Acceptable

⑧ L861Q (C8) 32.86 0.14 < 2 Acceptable



2) Profile of samples

① Non PNA mix

② G719X PNA mix

③ E19 del. PNA mix

④ T790M PNA mix

⑤ S768I(V2) PNA mix

⑥ E20 Ins. 3 dup PNA mix

⑦ L858R PNA mix

⑧ L861Q PNA mix

Table 8. Example of sample Ct values

Sample No.

Assay

Sample 1

Ct

Sample 2

Ct

Sample 3

Ct

Standard

Ct **ΔCt-2 *ΔCt-1

① Non PNA (S1) 27.71 27.06 30.75

② G719X (S2) 37.29 35.37 34.46 33.5 (⑨) ② - ① ⑨ - ②

③ E19 del. (S3) 35.79 30.22 38.00 34 (⑩) ③ - ① ⑩ - ③

④ T790M (S4) 35.03 36.34 35.81 33 (⑪) ④ - ① ⑪ - ④

⑤ S768I(V2) (S5) 34.33 34.07 33.10 33 (⑫) ⑤ - ① ⑫ - ⑤

⑥ E20 Ins. 3 dup (S6) 36.09 37.05 36.78 30 (⑬) ⑥ - ① ⑬ - ⑥

⑦ L858R (S7) 34.42 34.86 28.95 33 (⑭) ⑦ - ① ⑭ - ⑦

⑧ L861Q (S8) 38.37 38.00 35.76 33 (⑮) ⑧ - ① ⑮ - ⑧

*ΔCt-1 = [Standard Ct] – [Sample Ct or Clamping Control Ct]

**ΔCt-2 = [Sample Ct] – [Non PNA mix Ct (S1)]



Table 9. Analysis of data

Sample No.

Assay

Sample 1 Sample 2 Sample 3

ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1

② G719X (S2) 9.58 -3.79 8.31 -1.87 3.71 -0.96

③ E19 del. (S3) 8.08 -1.79 3.16 3.78 7.25 -4.00

④ T790M (S4) 7.32 -2.03 9.28 -3.34 5.06 -2.81

⑤ S768I(V2) (S5) 6.62 -1.33 7.01 -1.07 2.35 -0.10

⑥ E20 Ins. 3 dup (S6) 8.38 -6.09 9.99 -7.05 6.03 -6.78

⑦ L858R (S7) 6.71 -1.42 7.80 -1.86 -1.80 4.05

⑧ L861Q (S8) 10.66 -5.37 10.94 -5.00 5.01 -2.76

Results Wild E19 deletion L858R

1. When ΔCt-1 is equal to or greater than 2( ).

① ΔCt-2 is greater than 8, the sample is assessed to be wild.

② ΔCt-2 is equal to or less than 8( ), the sample is assessed to be mutated.

2. When △Ct-1 is greater than 0 and less than 2( ).





2. Using ABI 7900




Assay Clamping

control Ct ΔCt-1

Accep.

ΔCt-1 range Result

② G719X (C2) 34.40 -0.90 < 2 Acceptable


④ T790M (C4) 35.40 -2.40 < 2 Acceptable

⑤ S768I(V2) (C5) 31.90 1.10 < 2 Acceptable


⑦ L858R (C7) 34.84 -1.84 < 2 Acceptable

⑧ L861Q (C8) 36.22 -3.22 < 2 Acceptable




① Non PNA mix

② G719X PNA mix


④ T790M PNA mix



⑦ L858R PNA mix

⑧ L861Q PNA mix


Sample No.

Assay

Sample 1

Ct

Sample 2

Ct

Sample 3

Ct

Standard

Ct **ΔCt-2 *ΔCt-1

① Non PNA (S1) 28.69 28.35 25.16

② G719X (S2) 35.40 34.17 33.69 33.5 (⑨) ② - ① ⑨ - ②

③ E19 del. (S3) 37.82 35.51 35.60 34.5 (⑩) ③ - ① ⑩ - ③

④ T790M (S4) 36.12 35.45 35.68 33 (⑪) ④ - ① ⑪ - ④

⑤ S768I(V2) (S5) 33.23 33.16 33.16 33 (⑫) ⑤ - ① ⑫ - ⑤

⑥ E20 Ins. 3 dup (S6) 38.00 38.07 34.91 30 (⑬) ⑥ - ① ⑬ - ⑥

⑦ L858R (S7) 35.00 34.19 27.75 33 (⑭) ⑦ - ① ⑭ - ⑦

⑧ L861Q (S8) 39.06 37.82 36.45 33 (⑮) ⑧ - ① ⑮ - ⑧






Sample No.

Assay



② G719X (S2) 6.71 -1.90 5.82 -0.67 8.53 -0.19

③ E19 del. (S3) 9.13 -3.32 7.16 -1.01 10.44 -1.10

④ T790M (S4) 7.43 -3.12 7.10 -2.45 10.52 -2.68

⑤ S768I(V2) (S5) 4.54 -0.23 4.81 -0.16 8.00 -0.16

⑥ E20 Ins. 3 dup (S6) 9.31 -8.00 9.72 -8.07 9.75 -4.91

⑦ L858R (S7) 6.31 -2.00 5.84 -1.19 2.59 5.25

⑧ L861Q (S8) 10.37 -6.06 9.47 -4.82 11.29 -3.45

Results Wild Wild L858R









3. Using ABI QuantStudio 5




Assay Clamping

control Ct ΔCt-1

Accep.

ΔCt-1 range Result

② G719X (C2) 36.22 -2.72 < 2 Acceptable


④ T790M (C4) 35.52 -1.52 < 2 Acceptable

⑤ S768I(V2) (C5) 37.00 -4.00 < 2 Acceptable


⑦ L858R (C7) 34.55 -1.05 < 2 Acceptable

⑧ L861Q (C8) 35.97 -2.97 < 2 Acceptable




① Non PNA mix

② G719X PNA mix


④ T790M PNA mix



⑦ L858R PNA mix

⑧ L861Q PNA mix




Sample No.

Assay

Sample 1

Ct

Sample 2

Ct

Sample 3

Ct

Standard

Ct **ΔCt-2 *ΔCt-1

① Non PNA (S1) 22.05 23.30 23.23

② G719X (S2) 34.69 36.23 36.90 33.5 (⑨) ② - ① ⑨ - ②

③ E19 del. (S3) 35.84 29.26 36.41 34.5 (⑩) ③ - ① ⑩ - ③

④ T790M (S4) 35.66 35.03 35.03 34 (⑪) ④ - ① ⑪ - ④

⑤ S768I(V2) (S5) 34.38 36.27 35.78 33 (⑫) ⑤ - ① ⑫ - ⑤

⑥ E20 Ins. 3 dup (S6) 32.70 33.13 33.51 30 (⑬) ⑥ - ① ⑬ - ⑥

⑦ L858R (S7) 33.19 34.71 29.18 33.5 (⑭) ⑦ - ① ⑭ - ⑦

⑧ L861Q (S8) 35.68 35.97 37.20 33 (⑮) ⑧ - ① ⑮ - ⑧




Sample No.

Assay



② G719X (S2) 12.64 -1.19 12.92 -2.73 13.66 -3.40

③ E19 del. (S3) 13.79 -1.34 5.96 5.24 13.18 -1.91

④ T790M (S4) 13.61 -1.66 11.73 -1.03 11.80 -1.03

⑤ S768I(V2) (S5) 12.33 -1.38 12.97 -3.27 12.55 -2.78

⑥ E20 Ins. 3 dup (S6) 10.65 -2.70 9.83 -3.13 10.27 -3.51

⑦ L858R (S7) 11.14 0.31 11.40 -1.21 5.95 4.32

⑧ L861Q (S8) 13.63 -2.68 12.67 -2.97 13.96 -4.20

Results Wild E19 deletion L858R









QUALITY CONTROL

Each lot of PNAClamp™ EGFR Mutation Detection Kit (Ver.2) is tested against predetermined

specifications to ensure consistent product quality in accordance with PANAGENE’s ISO 9001 & 13485-

Certified Quality Management System

PERFORMANCE TEST

1. Analytical Sensitivity

The analytical sensitivity was determined by testing the standard EGFR mutant cell lines with the

PNAClamp™ EGFR Mutation Detection Kit (Ver.2).

The extracted DNA is measured as 25 ng and 50 ng. Each 25 ng and 50 ng DNAs were diluted to have 100,

10 and 1% of different mutant ratio. Three tests were performed with these 3 conditions of DNAs for 3

different batches of the kit.

The results showed that 1% mutation was detected for all cases the mutant DNA cell line concentrations.

If the ratio of mutant to wild type is 100-fold higher, 1% mutation can be detected. The detection ranged

from 10 to 10,000 copies, depending on the mutant location.

2. Analytical Specificity

The analytical specificity was determined by testing the wild cell lines without mutant DNA. Three tests

were performed on three batches of the kit using DNA (25, 50 and 100 ng) extracted from wild-type cell

lines (A549 and HeLa cells). The three tests showed wild-type locations. The tests of 25 and 50 ng DNA

from the mutated SW48, H1975, and PC9 cell lines showed wild-type locations, except for each mutant

location with no cross-reactivity.

3. Reproducibility

Experiments were performed to evaluate the reproducibility of three standard cell line DNAs, at 100, 10, 1

and 0% of different mutant ratio, for three batches, among three operators, for three days. PNAClamp™

EGFR Mutation Detection Kit (Ver.2) had a correct call rate of 100%. All the results showed little variation,

with %CV<5%.



REFERENCES

1. Kim et al., Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction

clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-

small cell lung cancer. Lung Cancer. 75 (3):321-325, 2012.

2. Han et al., Detection of EGFR Mutation Status in Lung Adenocarcinoma Specimens with Different

Proportions of Tumor Cells Using Two Methods of Differential Sensitivity. J Thorac Oncol. 7 (2):355-64,

2012.

3. Choi et al., PNA-mediated Real-Time PCR Clamping for Detection of EGFR Mutations. Bull. Korean

Chem. Soc. 31 (12): 3525-3529, 2010.

4. Lee et al., PNA-Mediated PCR Clamping for the Detection of EGFR Mutations in Non-Small Cell Lung

Cancer. Tuberc Respir Dis. 69:271-278, 2010

5. Noro et al., Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand

stimulation. BMC Cancer 6: 277, 2006.



EXPLANATION OF SYMBOLS ON THE LABEL

Batch Code

Use by (YYYY.MM.DD)

Manufacturer

EC Representative

In Vitro Diagnostic Medical Device

Catalog number

Temperature Limitation European conformity

PANAGENE Inc.


MT Promedt Consulting GmbH

Altenhofstrasse 80, 66386 St. Ingbert, Germany



ENDNOTES

The kit itself or any of the components in the kit cannot be modified, re-sold or transferred without the approval

of manufacturer. Information in this document is subject to change.

PANAGENE Inc.


Tel: +82-42-861-9296

Fax: +82-42-861-9297

E-mail: [email protected] (for general inquiry)

[email protected] (for order and quotation inquiry)

[email protected] (for technical inquiry)

Website: www.panagene.com

mailto:[email protected]%20(for



http://www.panagene.com/

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