QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
3 KRAS XNA mix #3 Codon 13 XNA and primers 1 x 200 µl 1 x 400 µl -20°C
4 KRAS XNA mix #4 Codon 61 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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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).
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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”.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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 #3 (S3) or
KRAS XNA Mix #4 (S4) or
KRAS XNA Mix #5 (S5) 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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
12
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 3 SAMPLE 4 SAMPLE 4 SAMPLE 4 SAMPLE 4 SAMPLE 4
SAMPLE 4
C
SAMPLE 5
SAMPLE 5
SAMPLE 5
SAMPLE 5
SAMPLE 5
SAMPLE 5 SAMPLE 6 SAMPLE 6 SAMPLE 6 SAMPLE 6 SAMPLE 6
SAMPLE 6
D
SAMPLE 7
SAMPLE 7
SAMPLE 7
SAMPLE 7
SAMPLE 7
SAMPLE 7 SAMPLE 8 SAMPLE 8 SAMPLE 8 SAMPLE 8 SAMPLE 8
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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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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
KRAS XNA mix #3 (C3) > 34
KRAS XNA mix #4 (C4) > 34
KRAS XNA mix #5 (C5) > 34
KRAS XNA mix #6 (C6) > 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:
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
16
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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
17
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.
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
18
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%
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
19
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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
20
Using Roche LC96 Profile of WT DNA Control
Profile of samples and controls
Understanding the Symbols
WT without
QClamping
WT with
Qclamping
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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)
Possible Cause Recommended Solutions
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
Possible Cause Recommended Solutions
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
Possible Cause Recommended Solutions
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
Possible Cause Recommended Solutions
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%
Possible Cause Recommended Solutions
Non-specific products Use melting curve analysis
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
23
PCR efficiency is <90%
Possible Cause Recommended Solutions
PCR inhibitors present in a reaction mixture
Re-purify template DNA
Non-uniform fluorescence intensity
Possible Cause Recommended Solutions
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
QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4)
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
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