qclamp™ kras codon specific mutation detection kit (exon 2 ... · the qclamp™ kras mutation...

QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)


Instruction Manual

Rev. 5.0

For Real-Time PCR Assays

# DC -10-0170 (30 samples)

# DC-10-0171 (60 samples)

Date of Revision: December 3, 2013

DOC-DC1100170_DC1100171

DiaCarta Inc.

3535 Breakwater Ave., Hayward, CA 94545

TEL: (510) 314-8858 FAX: (510) 735-8636

E-MAIL: [email protected]

MDSS GmbH

Schiffgraben 41

30175 Hannover,

Germany

mailto:[email protected]


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Contents

Components of the QClamp™ KRAS Mutation Detection Kit ............................................. 3

Storage Requirements ............................................................................................................... 3

Intended Use .............................................................................................................................. 4

KRAS Mutations and Cancer .................................................................................................. 4

QClamp Technology for Mutation Detection ...................................................................... 5

General Considerations ............................................................................................................ 6

Validated PCR Instruments for QClamp XNA Assays ......................................................... 7

Additional Equipment and Reagents Required .............................................................................. 8

Warnings and Precautions .................................................................................................................... 8

ASSAY PROCEDURE

Sample Preparation ....................................................................................................................... 9

DNA preparation from FFPE samples or solid tissue with QZol reagent

DNA preparation from cells with QZol reagent

Guidelines for using QZol Reagent on whole blood

Purified DNA sample (non-QZol)

Preparation and aliquoting of PCR mixes and samples ...................................................... 11

Set up master mixes for assays in 96-well plate, tube strips, or tubes

Dispense master mix, samples, and Clamping Controls

Real-Time PCR Reaction ....................................................................................................... 14

ANALYSIS OF RESULTS

Assessment of Real-Time PCR Results ................................................................................. 14

Clamping Controls (wild-type DNA control)

Judging validity of sample data based on non-XNA mix results

Judging validity of sample data based on Internal Control of HRM Curves

Scoring Detected Mutations ................................................................................................... 16

Assay Performance Characteristics ...................................................................................... 17

Symbols Used in Packaging.................................................................................................... 20

Ordering Information ............................................................................................................. 20

Troubleshooting ...................................................................................................................... 22

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


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KIT COMPONENTS

TABLE 1. COMPONENTS OF KITS #DC-10-0170 AND #DC-10-0171

No. Name of component Description Volume

(30 tests)

Volume

(60 tests)

Storage

1 Non XNA mix #1 Primers only 1 x 200 µl 1 x 400 µl -20°C

2 KRAS XNA mix #2 Codon 12 XNA and primers 1 x 200 µl 1 x 400 µl -20°C



5 KRAS XNA mix #5 Codon 117 XNA and primers

1 x 200 µl 1 x 500 µl -20°C

1 x 400 µl 1 x 500 µl -20°C

-20°C 1 x 500 µl -20°C

6 KRAS XNA mix #6 Codon 146 XNA and primers

1 x 200 µl 1 x 500 µl -20°C

1 x 400 µl 1 x 500 µl -20°C

-20°C 1 x 500 µl -20°C

7 KRAS XNA 2X premix PCR reaction premix 2 x 1.0 ml 4 x 1.0 ml -20°C

8 Clamping control Wild-type DNA 1 x 150 µl 1 x 300 µl -20°C

9 QZol Solution A Lysis Buffer A 2 x 1 ml 4 x 1 ml -20ºC

10 QZol Solution B Lysis Buffer B 2 x 1 ml 4 x 1 ml -20ºC

STORAGE REQUIREMENTS

The QClamp™ KRAS Codon-Specific Mutation Detection Kit (Exons 2, 3, and 4) should be stored at

-20 °C.


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INTENDED USE

The QClamp™ KRAS Mutation Detection Kit (Exon 2,3,4) is used to detect somatic mutations in

codon 12, codon 13, codon 61, codon 117 and codon 146 in the KRAS Proto-oncogene (Table 2) from

cell or tissues without DNA extractions. The kit is to be used by trained laboratory professionals, within

a laboratory environment, using (for example) fresh or formalin-fixed paraffin-embedded samples of

lung and colorectal biopsies and surgical tissue samples.

TABLE 2. KRAS MUTATIONS DETECTED BY THE KIT

Reagent Target Exon Amino Acid Change Nucleotide Change

KRAS Mutation 2 G12>A 35G>C

KRAS Mutation 2 G12>R 34G>C

KRAS Mutation 2 G12>D 35G>A

KRAS Mutation 2 G12>C 34G>T

KRAS Mutation 2 G12>S 34G>A

KRAS Mutation 2 G12>V 35G>T

KRAS Mutation 2 G13>D 38G>A

KRAS Mutation 2 G13>C 37G>T

KRAS Mutation 2 G13>R 37G>C

KRAS Mutation 3 Q61>K 181C>A

KRAS Mutation 3 Q61>L 182A>T

KRAS Mutation 3 Q61>R 182A>G

KRAS Mutation 3 Q61>H 183A>C

KRAS Mutation 4 K117>N 351A>C/T

KRAS Mutation 4 A146>T 436G>A

KRAS Mutation 4 A146>V 437C>T

KRAS MUTATIONS AND CANCER

The KRAS mutations are found in several cancers including colorectal, lung, thyroid, and pancreatic

cancers and cholangiocarcinoma. KRAS mutations are often located within codons 12 and 13 of exon

2, c o d o n 6 1 i n e x o n 3 a n d c o d o n s 1 1 7 a n d 1 4 6 i n E x o n 4 which may lead to abnormal

growth signaling by the p21- ras protein. These alterations in cell growth and division may trigger

cancer development as signaling is excessive. A KRAS mutation often serves as a useful prognostic

marker in drug response. For example, a KRAS mutation is considered to be a strong prognostic

marker of response to tyrosine kinase inhibitors such as gefitinib (Iressa) or erlotinib (Tarceva).


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KRAS mutations have a l s o been detected in many colorectal cancer patients and are associated with

responses to cetuximab (Erbitux) or panitumumab (Vectibix), which are used in colon cancer therapy.

The recent PEAK Phase 2 clinical study of Panitumumab and Bevacvizumab plus mFOLFOX6 for first

line treatment of metastatic colorectal cancer exemplified the need for extended testing of KRAS and

KRAS mutations at codons 61, 117 and 146 for selection of patients that would respond to anti-KRAS

antibody combination therapy.

QCLAMP™ TECHNOLOGY FOR MUTATION DETECTION

The QClamp™ KRAS Codon Specific Mutation Detection Kit is based on Xeno-Nucleic Acid

(XNA)-mediated PCR clamping technology. XNA is a synthetic DNA analog in which the

phosphodiester backbone has been replaced by a repeat formed by units of (2-aminoethyl)-glycine.

XNA-mediated PCR clamping relies on the following two unique properties of XNA probes:

First, XNA will hybridize tightly to its complementary DNA target sequence only if the sequence

is a complete match. When there is a mutation in the target gene, and therefore a mismatch is present,

the XNA:DNA duplex is unstable, allowing strand elongation by DNA polymerase.

Second, XNA oligomers are not recognized by DNA polymerases and cannot be utilized as primers

in subsequent real-time PCR reactions. Instead, the XNA oligomer serves as a sequence-selective

clamp to prevent amplification during subsequent PCR reactions.

The assay is sufficiently robust that conventional nucleic acid purification is not required. Tissue or

cells can be simply lysed with the QZol™ reagent provided, then an aliquot of this extract is added

directly to the PCR mixture containing DNA primers and the XNA “clamp”.


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FIGURE 1. PRINCIPLE OF THE QCLAMP™ KRAS CODON-SPECIFIC MUTATION DETECTION KIT

The QClamp XNA oligonucleotide binds the wild-type DNA near the hybridization site of the forward

PCR primer, thus blocking the action of the DNA polymerase. Genetic variations at the QClamp

binding site will prevent tight binding of the QClamp oligonucleotide, permitting the polymerase chain

reaction to produce a detectable amplicon.

GENERAL CONSIDERATIONS

Effective use of real-time PCR tests requires good laboratory practices, including maintenance of

equipment that is dedicated to molecular biology and is compliant with applicable regulations and

relevant standards. Use nuclease-free labware (pipets, pipet tips, reaction vials) and wear gloves when

performing the assay. Use fresh aerosol-resistant pipet tips for all pipetting steps to avoid cross

contamination of the samples and reagents.

Perform the QClamp assay protocol using only material (pipets, tips, etc.) dedicated to this application

in an area where no DNA matrixes (DNA, plasmid, or PCR products) have been introduced. Add

template DNA in a separate area (preferably a separate room) with material (pipets, tips, etc.) dedicated

only to this application. Use extreme caution to prevent DNase contamination that could result in

degradation of the template DNA, or DNA or PCR carryover contamination, which could result in a

false positive signal.

Reagents and instructions supplied in the kit have been tested for optimal performance. All reagents are

formulated specifically for use with this kit. Make no substitutions in order to ensure optimal


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performance of the kit. Further dilution of the reagents or alteration of incubation times and

temperatures may result in erroneous or discordant data.

VALIDATED REAL-TIME PCR INSTRUMENTS FOR QCLAMP XNA ASSAYS

The following instruments have been validated for use with QClamp XNA assays.

TABLE 3. REAL-TIME INSTRUMENTS TESTED WITH QCLAMP XNA ASSAYS

*Cepheid uses a 25 µl reaction volume. If using the Cepheid instrument, or for advice in optimizing

your protocol for other instruments, please contact DiaCarta.

Email: [email protected]

Tel: +1 510 314-8858

www.diacarta.com

Company Model

Bio-Rad CFX 96

Roche LightCycler LC96

Roche LightCycler 480 II

ABI ABI 7500

ABI ABI 7900

Qiagen Rotor-Gene Q

Cepheid* SmartCycler


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ADDITIONAL EQUIPMENT AND REAGENTS REQUIRED

Real-time PCR instrument capable of SYBR Green dye detection

0.2 ml DNase-free PCR tubes or plates

Pipettes (P-20, P-200, P-1000, P-200 multi-channel)

1.5ml microcentrifuge tubes

15 ml conical tubes

Microcentrifuge

Vortexer

PCR rack

Reagent reservoir

Distilled water

WARNINGS AND PRECAUTIONS

Use extreme caution to prevent contamination of PCR reactions with the Clamping Control.

Minimize exposure of the XNA 2X premix to room temperature for optimal amplification.

Avoid overexposing the XNA 2X premix solution to light for optimal fluorescent signal.

Use of non-recommended reagent volumes may result in a loss of performance and may also

decrease the reliability of the test results.

Use of non-recommended volumes and concentrations of the target DNA sample may result in a

loss of performance and may also decrease the reliability of the test results.

Use of non-recommended consumables with instruments may adversely affect test results.

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

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

Additional purification may be required if DNA has been extracted from a paraffin block.

Perform all experiments under proper sterile conditions using aseptic techniques.

Perform all procedures using universal precautions.

Wear personal protective apparel, including disposable gloves, throughout the assay procedure.

Do not eat, drink, smoke, or apply cosmetics in areas where reagents or specimens are handled.

Dispose of hazardous or biologically-contaminated materials according to the practices of your

institution.

Discard all materials in a safe and acceptable manner, in compliance with all legal requirements.

Dissolve reagents completely, then mix thoroughly by vortexing.

If exposure to skin or mucous membranes occurs, immediately wash the area with large amounts of

water. Seek medical advice immediately.

Do not use components beyond the expiration date printed on the kit boxes.

Do not mix reagents from different lots.

Return all components to the appropriate storage condition after preparing the working reagents.

Do not interchange vial or bottle caps, as cross-contamination may occur.


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ASSAY PROCEDURE

Step 1: Sample preparation (30 min) Lyse samples with QZol to release genomic DNA Step 2: Add QClamp Mixture (10min) Add lysates or clamp controls to QClamp mix (2X Premix and XNA Mix)

Step 3: Real-Time PCR Reaction (2 hours)

FIGURE 2. WORKFLOW OF THE QCLAMP KRAS CODON SPECIFIC MUTATION DETECTION KIT

Use standard pathology methodology to ensure specimen quality during collection, transport and

storage of samples. Alternate methodology for sample handling must be validated by the enduser.

1. SAMPLE PREPARATION

QZol™ Reagent is a complete and ready-to-use lysis reagent consisting of QZol Solutions A and B.

QZol releases genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin

into a form which can be used directly in PCR reactions without the need for DNA extraction. In

addition, the special properties of QZol„s chemistry can help keep DNA in linear format to optimize

hybridization and increase PCR efficiency.

Typical sources of genomic DNA for mutation detection by the kit include sections of formalin-fixed,

paraffin-embedded (FFPE) samples, as well as fresh or frozen tissue from surgical procedures and

biopsies. QZol has been validated on these sample types, as well as on cultured cells and cells purified

from blood such as peripheral blood lymphocytes, polynuclear cells, and granulocytes. An extraction

procedure for whole blood with the reagent has not yet been validated, but should work with some

optimization depending on coagulant used, etc. Contact DiaCarta at [email protected] for

assistance with genomic DNA protocol optimization.

Other methods for purifying genomic DNA, such as homebrew methods or commercially-available

products, will also work with the kit. Regardless of which approach is used, use the same cellular

fraction and DNA extraction method each time the assay is performed.

http://www.lifetechnologies.com/order/catalog/product/10503027



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DNA preparation from FFPE samples or solid tissue with QZol reagent

1. Thaw QZol Solution A and QZol Solution B at room temperature or in a water bath

2. Place approximately 50 µl of a 10 µm FFPE section or the same amount of fresh or frozen solid tissue

into a microcentrifuge tube. Ratio of solid sample to reagent volume should be roughly 1:1.

3. Add 50 µl of QZol Solution A to each tube.

4. Place sample tubes into a heating block at 95 °C for 5 min, long enough to melt the paraffin.

5. Remove and vortex the sample tube for 10 seconds.

6. Return the tubes to the 95 °C block for 20 minutes, removing every 5 minutes to vortex 10 seconds.

7. Remove sample tubes from heating block and add an equivalent volume of QZol Solution B as was

added of QZol Solution A. If 50 µL of Solution A was added earlier, now add 50 µl of Solution B.

8. Vortex each sample for 10 seconds.

9. Spin down the sample preparation tube for 30 seconds in a microcentrifuge.

10. Collect the supernatant, the QZol lysate, avoiding the pellet, for use in PCR procedure, cool to room

temperature.

DNA preparation from cells with QZol reagent

For softer and moister tissues such as cultured cells or cells purified from blood such as peripheral blood

lymphocytes, polynuclear cells, and granulocytes, modify the protocol to add twice the volume of QZol

reagent as sample volume.

1. Thaw QZol Solution A and QZol Solution B at room temperature or in a water bath

2. Add anywhere from 200 to 100,000 cells to a microcentrifuge tube, ideally a sample volume of

approximately 50 µl. The sample to reagent volume ratio for these tissue types should be roughly 1:2.

3. Add 100 µl of QZol Solution A to each tube and vortex for 10 seconds.

4. Place sample tubes into a heating block at 95 °C for 20 minutes, removing every 5 minutes to vortex

10 seconds.

5. Remove sample tubes from heating block and add an equivalent volume of QZol Solution B as was

added of QZol Solution A. If 100 µL of Solution A was added earlier, now add 100 µl of Solution B.

6. Vortex each sample for 10 seconds.

7. Spin down the sample preparation tubes for 30 seconds in a microcentrifuge.

8. Collect the supernatant, the QZol lysate, avoiding the pellet, for use in PCR procedure, cool to room

temperature.

Guidelines for using QZol Reagent on Whole Blood

A generalized extraction procedure for QZol Reagent on whole blood has not been established. Whole

blood is a complex tissue and different coagulation reagents produce final products with varying


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characteristics. However, a reasonable starting point would be the incubation protocol for cells on a

sample size of 200 µl whole blood along with 400 µl each of Solution A and Solution B. Contact

DiaCarta at [email protected] for assistance with whole blood protocol optimization.

Purified DNA Sample (non-QZol)

The QClamp real-time PCR reaction is optimized for DNA samples containing 5-10 ng of purified

genomic DNA. If you are working with samples consisting of purified DNA, dilute the DNA to a

concentration of 5 ng/μl in 1X TE buffer at pH 8.0. Store samples at +4 to +8 °C for short periods, up to

one week. Store at –20 °C if longer-term storage is required.

2. PREPARATION AND ALIQUOTING OF PCR MIXES AND SAMPLES

Each sample of potentially mutant DNA requires one reaction for each mutation site detected by the kit,

plus an XNA-free control. The XNA-free control insures that the supplied primers and polymerase are

working properly on the sample. The KRAS Codon-Specific kit detects the mutation sites listed in

Table 2, therefore a total of six reactions will be required for each sample.

A set of Clamping Controls must also be run every time the assay is run. Clamping Controls use

wild-type DNA as the sample. Wild-type DNA should have no mutations, therefore the XNA probes

will bind strongly, blocking the polymerase from making amplicons. However, non-XNA mix #1 with

the Clamping Control should make amplicons efficiently, providing another way to monitor

performance of the primers, polymerase, and sample.

Each kit contains enough material to run five sets (30-sample test kit) or ten sets (60-sample test kit) of

Clamping Controls, or one Clamping Control set for every six samples. Further quantities of KRAS

wild-type genomic reference DNA control can be purchased as a separate item, if desired.

Depending on how many samples will be processed in a given experiment, different strategies are used

for creating master mixes. The most typical application involves testing in 96-well plates, but the assay

can also be run in tube strips or individual tubes.

The QClamp XNA real-time assay protocol uses 20 μl reaction volumes. Each reaction will contain 10

μl 2X Premix, 6 μl of one of four XNA Mixes, and 4 μl of sample, for a total of 20μl.

Adjust amounts appropriately for different reaction volumes.



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TABLE 4. COMPONENTS OF THE QCLAMP XNA ASSAY REACTION VOLUME

Components Volume

KRAS XNA 2X Premix 10 μl

XNA Mix (#1, #2, #3, #4, #5, or #6) 6 μl

DNA sample or Clamping Control 4 μl

Total volume 20 μl

Set up master mixes for assays in 96-well plate, tube strips, or tubes

(This is a suggested method. Other approaches can achieve the final result.)

1. Consecutively label six tubes as M1, M2, M3, M4, M5, M6.

These will be master mix tubes containing XNA Mixes #1, #2, #3, #4, #5, and #6 respectively, plus the

2X PreMix (containing polymerase, SYBR Green and appropriate buffers).

Tip: It is good practice to go 10% over when putting master mixes together to insure not running out of

master mix prematurely when aliquotting.

2. Add the appropriate amount of 2X Premix and XNA Mix to its M tube. See table 5 for

appropriate volumes.

TABLE 5. SETTING UP PCR REACTION MASTER MIXES

Table 5 is based on the following calculations to determine the number of microliters of each XNA Mix

and 2X Premix to aliquot to its respective master mix tube, where N = total number of samples. For

sample amounts not indicated in Table 5, the following calculations may be used:

(N + 2) x 6 = Microliters of XNA Mix for each master mix tube of the corresponding number.

(N+2) x 10 = Microliters of 2X Premix in every master mix tube.

(The N+2 calculation is to account for the Clamping Controls and overage.)

# samples

(Volume preparation

includes amount

required for Clamping

Control tube)

Non-XNA Mix #1 (S1) or

KRAS XNA Mix #2 (S2) or




KRAS XNA Mix #6 (S6)

KRAS XNA 2X

Premix

Total Volume Sample volume

10 72 µl 120 µl 192 µl Split 16 µl to each

tube then add 4 µl of

sample to appropriate

tubes

20 132 µl 220 µl 352 µl

30 172 µl 320 µl 492 µl

40 252 µl 420 µl 672 µl

50 312µl 520 µl 832 µl

60 372 µl 620 µl 992 µl


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Dispense master mix, samples, and Clamping Control

3. Dispense 16 µl master mix across a 96-well plate or into tubes

Transferring the contents of each master mix tube to a reagent reservoir now enables use of a

multichannel pipettor in dispensing across a plate or into strips.

Alternatively, a repeating pipettor would be useful for an individual tubes assay format.

In the case of 96-well plates, the exact plate layout for the next step can be set to the user‟s preference.

However, take care to remember which wells are for which XNA Mixes, to insure that all potential

detected mutations and XNA minus controls are processed properly.

A suggested layout involves using a 8-channel pipettor to pipet 16 µl of master mix into the columns

on the plate, that is, to pipet 16 µl of M1 into columns 1 and 7, 16 µl of M2 in columns 2, 8, and so on.

6 ul NonXNA Mix #1

6 ul XNA

Mix #2

6 ul XNA

Mix #3

6 ul XNA

Mix #4

6 ul XNA Mix

#5

6 ul XNA Mix

#6

6 ul NonXNA Mix #1

6 ul XNA Mix #2

6 ul XNA Mix #3

6 ul XNA Mix

#4

6 ul XNA Mix #5

6 ul

XNA Mix #6

1 2 3 4 5 6 7 8 9 10 11

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A

SAMPLE 1

SAMPLE 1

SAMPLE 1

SAMPLE 1

SAMPLE 1

SAMPLE 1 SAMPLE 2 SAMPLE 2 SAMPLE 2 SAMPLE 2 SAMPLE 2

SAMPLE 2

B

SAMPLE 3

SAMPLE 3

SAMPLE 3

SAMPLE 3

SAMPLE 3


SAMPLE 4

C

SAMPLE 5

SAMPLE 5

SAMPLE 5

SAMPLE 5

SAMPLE 5


SAMPLE 6

D

SAMPLE 7

SAMPLE 7

SAMPLE 7

SAMPLE 7

SAMPLE 7


SAMPLE 8

E

SAMPLE 9

SAMPLE 9

SAMPLE 9

SAMPLE 9

SAMPLE 9

SAMPLE 9 SAMPLE

10 SAMPLE

10 SAMPLE

10 SAMPLE

10 SAMPLE

10

SAMPLE

10

F

SAMPLE 11

SAMPLE 11

SAMPLE 11

SAMPLE 11

SAMPLE 11

SAMPLE 11

SAMPLE 12

SAMPLE 12

SAMPLE 12

SAMPLE 12

SAMPLE 12

SAMPLE

12

G

SAMPLE 13

SAMPLE 13

SAMPLE 13

SAMPLE 13

SAMPLE 13

SAMPLE 13

SAMPLE 14

SAMPLE 14

SAMPLE 14

SAMPLE 14

SAMPLE 14

SAMPLE

14

H

SAMPLE 15

SAMPLE 15

SAMPLE 15

SAMPLE 15

SAMPLE 15

SAMPLE 15

CLAMPING CONTROL

CLAMPING CONTROL

CLAMPING CONTROL

CLAMPING CONTROL

CLAMPING CONTROL

CLAMPING CONTROL

10 ul 2X Premix, all wells

FIGURE 3. SUGGESTED PLATE LAYOUT

4A. Dispense 4 µl of sample DNA and Clamping Control DNA into wells

With a plate layout as described in Figure 3, where each column represents a different XNA Mix, use six pipet

tips on an 8-channel pipettor or a repeating pipettor with a single tip to pipet 4 µl of Sample 1 into each of the


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first six wells of Row A, then 4 µl of Sample 2 into each of the next six wells in that row, and so on, until all

samples are loaded.

Pipet the clamping controls into the last six wells of Row H.

4B. Dispense 4 µl of sample DNA and Clamping Control DNA into tubes

If pipetting samples into tubes instead of 96-well plates, label tubes as “S” followed by the number of the mix (1,

2, 3, 4, 5, or 6), a hyphen, then the sample number. For example, if running 15 samples, label tubes as S1-1, S1-2

…..S1-15. Repeat for S2, S3, S4, S5, and S6. Label the single set of Clamping Control reaction tubes as C1, C2,

C3, C4, C5, C6.

When all reagents have been loaded, tightly close the PCR tubes or seal the 96-well plate to prevent evaporation.

3. REAL-TIME PCR REACTION

Set up the real-time PCR instrument to read SYBR Green at 60 °C. Perform real-time PCR using the cycling

conditions described below.

TABLE 6. CYCLING CONDITIONS FOR QCLAMP XNA ASSAYS

One cycle

Pre-denaturation 95oC 5 minutes

Four-step cycling (40 cycles total)

Denaturation 95oC 20 seconds

Qclamping 70oC 20 seconds

Primer Annealing 60oC 30 seconds

Extension* 60oC 30 seconds

ANALYSIS OF RESULTS

ASSESSMENT OF REAL-TIME PCR RESULTS

Determine the Cq value for each PCR reaction. Cq is the cycle threshold, the cycle number at which a signal

is detected above background fluorescence. The lower the cycle number at which signal rises above

background, the stronger the PCR reaction it represents (**please see MIQE Guidelines under References).

Clamping Controls (wild-type DNA control)

The Cq values of the Clamping Controls (tubes C1-C6) should fall in the range given in the table below. These

values are expected because the combination of wild-type DNA with XNA probes in C2, C3, C4, C5, and C6

should block amplification, while the absence of probes in C1 would produce a robust level of amplification.

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


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TABLE 7. ACCEPTABLE CQ RANGES FOR THE CLAMPING CONTROLS

Assay Acceptable Cq Range

Non-XNA mix #1 (C1) 23 ≤ Cq ≤ 30

KRAS XNA mix #2 (C2) > 34





Judging validity of sample data based on non-XNA mix results

In considering the Cq values for each sample (S1-S6), note that the Cq values of any non-XNA mix #1

reaction should be in the range of 22-34. The Cq value of non-XNA mix #1 (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 Cq value of the non-XNA mix #1 (S1).

TABLE 8. NON-XNA CONTROLS FOR SAMPLE PURITY AND CONCENTRATION

Validity Cq value of

non-XNA mix #1 Descriptions and recommendations

Optimal 23 < Cq < 30 The amplification and amount of DNA sample were

optimal.

Acceptable 30 < Cq < 34 The target gene was amplified at low efficiency. For a more

reliable result, repeat the PCR reaction with more DNA.

Invalid Cq ≤22 Possibility of a false positive is high. Repeat the PCR

reaction with less DNA.

Invalid Cq ≥ 34 The amplification has failed. Check DNA amount and

purity. A new DNA prep may be required.

Judging validity of sample data based on Internal Control of HRM curves

If test sample is negative, please check the HRM melting profile derivative plots ( -dF/dT against

T) to make sure it is true negative.

1. The -dF/dT should be 0.10 or higher

2. If the -dF/dT is less than 0.10, PCR reaction is inhibited, the obtained data must be

discarded and the experiment should be repeated.

Normal PCR reaction HRM profiles:


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Sample 1

-dF/dT > 0.3

Sample 2

-dF/dT > 0.3

-dF/dT = < 0.10 PCR reaction inhibited.

SCORING DETECTED MUTATIONS

After measuring and recording the Cq values for all six reactions of each sample and all six Clamping

Control reactions, next determine ΔCq values for all the mutation sites in every sample.

Subtract the Cq of each sample that contained XNA Mix#2 from the Cq of Clamping Control 2 to get

that set of ΔCq values, then subtract the Cq of each sample that contained XNA Mix #3 from the Cq of

Clamping Control 3, then subtract the Cq of each sample that contained XNA Mix #4 from the Cq of

Clamping Control 4, then subtract the Cq of each sample that contained XNA Mix #5 from the Cq of

Clamping Control 5, and finally subtract the Cq of each sample that contained XNA Mix #6 from the

Cq of Clamping Control 6.


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Mutated samples are defined by conditions where the mutated allele yields Cq < 40, and the ΔCq

relative to the Clamped Control using the same XNA probe > 1.5. For example, if the Cq of the C2

Clamping Control is 39 and the Cq of sample S1-2 is 35, then the ΔCq = 39 - 35 = 4.0, or >1.5, so the

sample is scored positive for a mutation.

If performing sample replicates, calculate the mean ΔCq for each sample, the standard deviation (SD),

and the mutation threshold (MT) value, where MT = mean ΔCq−2SD.

ASSAY PERFORMANCE CHARACTERISTICS Analytical performance

The specific performance characteristics of the QClamp KRAS Mutation Detection kit were

determined by studies involving KRAS-defined genomic DNA reference samples obtained from

Horizon Diagnostics (Cambridge, England). These samples have been characterized genetically as

containing heterozygous mutations in the coding sequence of the KRAS gene at codons 12, 13, 61, 117,

and 146. These single nucleotide polymorphisms in the KRAS gene have been confirmed by droplet

digital PCR (ddPCR) and genomic DNA sequencing. Additional samples consisted of formalin-fixed,

paraffin-embedded (FFPE) reference and patient tissue samples, as well as KRAS wild-type DNA (no

mutations).

Analytical accuracy and comparison to reference method

QClamp analytical accuracy is verified and validated through testing of samples with known

mutations. Sample mutation status was verified through sequencing. Three studies were done to

demonstrate concordance in mutation status of FFPE samples tested with QClamp Mutation Detection

Kit relative to sequencing. A set of sample were chosen for evaluation based on mutation status. In a

blinded manner, samples were chosen to be tested with QClamp Mutation Detection Kit to be

compared to mutation status returned from sequencing. The results demonstrated that the QClamp

Mutation Detection Kit reported 100% match to sequencing. The results are confirmed by performance

from three different test sites and three different sets of clinical samples.

Cut-off

Along with studies for analytical accuracy, FFPE samples were tested to establish cut-off for the assay.

Cut-off for positive mutation has been established at ΔCq > 1.5.

Interfering substances

A study was performed to evaluate the impact of potentially interfering substances on the performance

of the QClamp KRAS Mutation Detection Kit. Potentially interfering substances tested were paraffin,

ethanol, QZol Solutions A and B, and Proteinase K. The impact of each substance on resultant ΔCq and

mutation status of test samples was determined via spiking experiments conducted at three different

concentrations, 0.1%, 1% and 5%. None of the potentially interfering substances evaluated at

concentrations encountered in normal use impacted the ability of the QClamp KRAS Mutation

Detection Kit to distinguish between mutation-positive and mutation-negative samples.


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Multiple freeze/thaw cycles

The effect of 1, 3, 5, and 8 freeze-thaw cycles were tested in QClamp KRAS Mutation Detection Kit

reagents. There is no effect up to 5 freeze-thaw cycles on the QClamp KRAS Mutation Detection Kit to

distinguish between mutation positive and mutation negative samples. Caution: Repeated freeze-thaw

cycles may decrease the reliability of test results.

Shelf-life

6 months after kit is open; 1 year after receiving for unopened kit.

Repeatability and reproducibility

The precision of QClamp KRAS Mutation Detection Kit was determined with defined analyte levels

of mutated DNA. To establish lot to lot variation, a reproducibility study of QClamp Mutation

Detection was performed using three different kit lots. Each lot was tested on three separate dates

testing one wild-type and one sample for each mutation with the QClamp KRAS Mutation Detection

Kit. Inter-assay %CV was established using the same lot of reagents tested by three different users,

performed at three different sites, with tests run one-two times a day for three days. Intra-assay %CV

was established through performance of QClamp Mutation Detection Kit with samples run in

triplicate and repeated for three days. All testing was done using sequence verified samples from

Horizon Diagnostics. Reproducibility is demonstrated based on %CV of Cq values with a rate of

100% correct mutation calls for all assays across multiple lots and operators for both within and

between laboratory experiments.

TABLE 9. REPRODUCIBILITY RESULTS SUMMARY

Variation %CV

Intra-assay ≤ 3%

Inter-assay ≤ 5%

Lot-to-lot variation ≤ 3%


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Limit of Detection

To determine the limit of detection (LOD) for the kit, a QClamp assay was run using a serial dilution of

mutant DNA in wild-type background. Mutant samples were sequence verified by Horizon

Diagnostics. Mutant concentrations tested were 50, 10, 5, 1, and 0.1%. Results demonstrate effective

clamping of wild type, providing reproducible detection of mutations at concentrations as low as 0.1%.

Mutant Dilution Study:

ΔCq 0.1% Mutant = 1.65 ΔCq = Cq of negative control – Cq of sample

ΔCq 1.0 % Mutant = 3.0 ΔCq = Cq of negative control – Cq of sample


20

Using Roche LC96 Profile of WT DNA Control

Profile of samples and controls

Understanding the Symbols

WT without

QClamping

WT with

Qclamping


21

SYMBOLS USED IN PACKAGING

TABLE 10. SYMBOLS USED IN PACKAGING

ORDERING INFORMATION: TABLE 11. ORDERING INFORMATION

QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2,3,4)

Symbol Definition

In vitro diagnostic device

Catalog number

Manufactured by

Temperature limitation

Batch code

Use by date

Authorized representative in the

European Community

CE Mark

2012-11-25 Date format (year-month-day)

2012-11 Date format (year-month)

Product Name Cat.Number Size Reader

Platform KRAS Mutations

QClamp™ KRAS Codon Specific

Mutation Detection Kit (Exon 2, 3,

4)

DC-10-0170 30

samples

Real-time

PCR

Analysis

Mutations in Exon 2 codon 12,

codon 13, Exon 3 codon 61 and Exon

4 codons 117 and 146

QClamp™ KRAS Codon Specific

Mutation Detection Kit (Exon 2, 3,

4)

DC-10-0171 60

samples

Real-time

PCR

Analysis

Mutations in Exon 2 codon 12,

codon 13, Exon 3 codon 61 and Exon

4 codons 117 and 146


22

TROUBLESHOOTING:

Negative result for Clamping Control with Non-XNA Mix #1

Possible Cause Recommended Solutions

Pipetting error Check pipetting scheme and setup of the reaction. Repeat the PCR run

Inappropriate storage of kit components

Store all kit components at appropriate temperature according to label, also see Kit Components Table (Table 1)

No signal (even in Clamping Controls with Non-XNA Mix #1)


Pipetting error or omitted reagents Check pipetting scheme and the setup of the reaction. Repeat the PCR run

Inhibitory effects of the sample material, caused by insufficient purification

Repeat the RNA preparation.

See manual section “Judging validity of sample data based on

Internal Control of HRM curves”

Fluorescence intensity too low


Inappropriate storage of kit components

Store all kit components at appropriate temperature according to label, also see Kit Components Table (Table 1)

Very low initial amount of target DNA

Increase the amount of sample DNA (Depending on chosen method of DNA preparation, inhibitory effects may occur)

No amplification curve and no PCR product visible on a gel


PCR inhibitors present in the reaction mixture

Re-purify template DNA

Inhibition by excess volume of the RT reaction

Volume of the RT reaction product added to qPCR reaction should not exceed 10% of the total qPCR reaction volume

Pipetting error or missing reagent Repeat the PCR reaction; check the concentrations of template and primers; ensure proper storage conditions of all reagents

Annealing temperature is not optimal

Optimize the annealing temperature in 3°C increments

No amplification curve but PCR product visible on a gel


qPCR instrument settings are incorrect

Check if instrument settings are correct (dye selection, reference dye, filters)

Inactive fluorescence detection Fluorescent detection should be activated and set at extension or annealing/extension step of the thermal cycling protocol

Instrument problems Refer to the instrument manual for troubleshooting

PCR efficiency is >110%


Non-specific products Use melting curve analysis


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PCR efficiency is <90%


PCR inhibitors present in a reaction mixture

Re-purify template DNA

Non-uniform fluorescence intensity


Contamination of the thermal cycler Perform decontamination of your real-time cycler according to the supplier‟s instructions

Poor calibration of the thermal cycler Perform calibration of the real-time cycler according to the supplier‟s instructions


24

REFERENCES

1. Beau-Faller et al., Detection of K-Ras mutations in tumour samples of patients with

non-small cell lung cancer using PNA-mediated PCR clamping. Br J Cancer. 2009

Mar 24;100(6):985-92.

2. Chang et al., Fast simultaneous detection of K-RAS mutations in colorectal cancer.

BMC Cancer. 2009 Jun 11;9:179.

3. Jeong et al., Rapid and Sensitive Detection of KRAS Mutation by Peptide Nucleic Acid based Real-time PCR Clamping: A Comparison with Direct Sequencing between Fresh Tissue and Formalin-fixed and Paraffin Embedded Tissue of Colorectal Cancer. The Korean Journal of Pathology 2011; 45: 151-169

4. Kobunai et al., The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology : A comparison between direct sequencing and real-time PCR. Biochem Biophys Res Commun. 2010 Apr 23;395(1):158-62.

5. Kwon et al., Frequency of KRAS, KRAS, and KRAS mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR and direct sequencing in formalin-fixed, paraffin-embedded tissue. Pathol Res Pract. 2011 Dec 15;207(12):762-8.

6. Ørum, Henrik., PCR Clamping.. Curr. Issues Mol. Biol. 2000; 2(1), 27-30.

7. Powell et. al., Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping. Analytical Biochemistry 1998; 260: 142–8.

8. Schwartzberg et. al., Analysis of KRAS/KRAS mutations in PEAK: A randomized phase 2 study of FOLFOX6 plus panitumumab (pmab) or bevacizumab as first-line treatment for wild-type KRAS (exon 2) metastatic colorectal cancer (mCRC). ASCO Meeting 2013, J Clin Oncol 31, 2013 (suppl; abstr 3631).

9. **MIQE Reference: "The MIQE Guidelines: Minimum Information for Publication

of Quantitative Real-Time PCR Experiments". Stephen A. Bustin et. al., Clin Chem. 55

(4): 611–22 (2009). http://www.clinchem.org/content/55/4/611

DiaCarta Inc.

3535 Breakwater Ave.

Hayward, CA94545 Email: [email protected]

Tel: +1 510 314-8858

Fax:+1 510 735 8636

www.diacarta.com

http://www.clinchem.org/content/55/4/611

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