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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection
Kit
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation
Detection Kit
Instruction Manual
Rev. 8.0
For Real-Time PCR Assays
# DC-10-0814 (30 samples)
For research use only
Instruction Version: Rev. 8.0
Date of Revision: April 2014
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
Contents
Components of the QClamp™ BRAF Mutation Detection Kit............................................. 3
Storage Requirements............................................................................................................... 3
Intended Use.............................................................................................................................. 4
BRAF Mutations and Cancer.................................................................................................. 4
QClamp Technology for Mutation Detection......................................................................... 4
Liquid Biopsy Samples............................................................................................................. 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
Guidelines for using QZol Reagent on whole blood
Purified DNA sample (non-QZol)
Preparation and aliquoting of PCR mixes and samples...................................................... 10
Set up mastermixes for assays in 96-well plate, tube strips, or tubes
Dispense master mix, samples, and Clamping Controls
Real-Time PCR Reaction....................................................................................................... 13
ANALYSIS OF RESULTS
Assessment of Real-Time PCR Results..................................................................................13
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................................................................................................... 15
Assay Performance Characteristics.......................................................................................15
Symbols Used in Packaging....................................................................................................20
Ordering Information.............................................................................................................20
Troubleshooting...................................................................................................................... 21
References ................................................................................................................................23
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
KIT COMPONENTS
TABLE 1. COMPONENTS OF KITS #DC-10-0814
No.
Name of component
Description
Volume
Storage
(30 tests)
1
Non XNA mix #1
Primers only
1 x 200 µl
-20°C
2
BRAF XNA mix #2
Codon 600 XNA and primers
1 x 200 µl
-20°C
3
BRAF XNA 2X premix
PCR reaction premix
1 x 0.8 ml
-20°C
4
Clamping control
Wild-type DNA
1 x 88 µl
-20°C
5
QZol Solution A
Lysis Buffer A
2 x 1.0 ml
-20°C
6
QZol Solution B
Lysis Buffer B
2 x 1.0 ml
-20°C
STORAGE REQUIREMENTS
The QClamp™ BRAF Liquid Biopsy Codon-Specific Mutation Detection Kit should be
stored at -20 °C.
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
INTENDED USE
This kit is for research use only.
The QClamp™ BRAF Liquid Biopsy Codon Specific Mutation Detection Kit is used to detect 5
somatic mutations in codon 600 in the BRAF serine/threonine protein kinase gene (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
malignant melanoma, colorectal cancer, lung cancer and papillary thyroid cancer biopsies and surgical
tissue samples.
TABLE 2. BRAF MUTATIONS DETECTED BY THE KIT
Reagent Target
Exon
Amino Acid Change
Nucleotide Change
BRAFMutation
15
V600>E
c1799T>A
BRAF Mutation
15
V600>K
c1798_1799GT>AA
BRAF Mutation
15
V600>D
c1799_1800TG>AT
BRAF Mutation
15
V600>R
c1798_1799GT>AG
BRAF Mutation
15
V600>M
c1798G>A
BRAF MUTATIONS AND CANCER
The B-type Raf Kinase (BRAF) protein is a serine/threonine kinase that has important roles in
regulating the MAP kinase/ERK signaling pathways, affecting cellular proliferation,
differentiation, and programmed cell death. A BRAF mutation is commonly found in many
human cancers including melanoma, colorectal cancer, lung cancer, and papillary thyroid
carcinoma. The most common mutations in BRAF occur in codon 600, where an amino acid
substitution in the activation segment of the kinase domain creates a constitutively active form
of the protein. The V600E and V600K mutations are found in high frequencies in human cancer
V600E 70-90% and V600K 10-15%. BRAF mutations are generally found in tumors that are
wild-type for BRAF, BRAF and BRAF. Therefore, BRAF V600E mutation may serve as a
biomarker for diagnosis, prognosis, and treatment options for cancer patients.
QCLAMP™ TECHNOLOGY FOR MUTATION DETECTION
The QClamp™ Liquid Bi opsy BRAF 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:
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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”.
FIGURE 1. PRINCIPLE OF THE QC LAMP ™ BRAF C ODON -S PECIFIC MUTATION DETECTION K IT
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.
LIQUID BIOPSY SAMPLES
QClamp ‘liquid biopsy’ DNA blood tests provide precise insight into the genetics of solid tumors. This
is accomplished with highly sensitive real-time QClamp PCR technology. With a liquid biopsy,
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
treatment decisions can now be made with the right information at any time. Non-invasive. Sensitive.
Real-time.
Distant metastases harbor unique genomic characteristics not detectable in the corresponding primary
tumor of the same patient and metastases located at different sites show a considerable intra-patient
heterogeneity. Thus, the mere analysis of the resected primary tumor alone (current standard practice in
oncology) or, if possible, even reevaluation of tumor characteristics based on the biopsy of the most
accessible metastasis may not reveal sufficient information for treatment decisions. This dilemma can
be solved by a new diagnostic concept: liquid biopsy, that is, analysis of therapeutic targets and drug
resistance–conferring gene mutations on circulating tumor cells (CTC) and cell-free circulating tumor
DNA (ctDNA) released into the peripheral blood from metastatic deposits.
QClamp liquid biopsy technology (QLBT) enables CTCs and ctDNA to be effective biomarkers in
clinical oncology. Both CTCs and ctDNA are interesting complementary technologies that can be used
in parallel in future trials assessing new drugs or drug combinations. DiaCarta’s QClamp liquid biopsy
products will contribute to a better understanding and clinical management of drug resistance in
patients with cancer.
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
performance of the kit. Further dilution of the reagents or alteration of incubation times and
temperatures may result in erroneous or discordant data.
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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
Company
Bio-Rad
Roche
Roche
ABI
ABI
Qiagen
Cepheid*
Model
CFX 96
LightCycler LC96
LightCycler 480 II
ABI 7500
ABI 7900
Rotor-Gene Q
SmartCycler
*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: information@diacarta.com
Tel: +1 510 314-8858
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
www.diacarta.com
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
ASSAY PROCEDURE
Step 1: Sample preparation (20 min)
Lyse samples with QZol to release genomic DNA
Step 2: Add QClamp Mixture
Add lysates or clamp controls to QClamp mix (2X Premix and XNA Mix)
Step 3: Real-Time PCR Reaction (average 90 min)
FIGURE 2.W ORKFLOW OF THE QC LAMP BRAF M UTATION 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.
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.
CAUTION: Use only whole blood treated with disodium ethylenediaminetetraacetate (EDTA).
Whole blood treated with heparin will not work well with this test.
DNA Preparation using QZol Reagent on Whole Blood
1. Thaw QZol Solution A and QZol Solution B at room temperature or in a water bath
2. Add anywhere from 50 µl of Whole Blood to a microcentrifuge tube. The sample reagent volume
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
ratio for whole blood should be roughly 1:2.
3. For every 50 µL of cells, 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 100 µl of QZol 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.
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 BRAF Codon-Specific kit detects the mutation sites listed in Table 2,
therefore a total of two 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) of Clamping Controls, or one
Clamping Control set for every six samples. Further quantities of BRAF 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.
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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 each XNA Mix, and 4 μl of sample, for a total of 20μl.
Adjust amounts appropriately for different reaction volumes.
TABLE 4. COMPONENTS OF THE QCLAMP XNA ASSAY REACTION VOLUME
Components
Volume
BRAF XNA 2X Premix
10 μl
XNA Mix (#1 or #2)
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 two tubes as M1 or M2.
These will be master mix tubes containing XNA Mixes #1 and #2 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
# samples
Non-XNA Mix #1 (S1)
or BRAF XNA Mix #2
(S2)
BRAF XNA 2X
Premix
Total Volume
Sample volume
10
72 µ l
120 µ l
192 µ l
20
132 µ l
220 µ l
352 µ l
Split 16 µ l to each tube
then add 4 µ l of sample
to appropriate tubes
30
172 µ l
320 µ l
492 µ l
40
252 µ l
420 µ l
672 µ l
50
31 2 µ l
520 µ l
832 µ l
60
372 µ l
620 µ l
992 µ l
(Volume preparation
includes amount
required for Clamping
Control tube)
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:
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
(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.)
Dispense master mix, samples, and Clamping Controls
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, 3, 5, 7, and 16 µl of M2 in columns 2, 4, 6, 8.
A
B
C
D
E
F
G
H
6 ul
Non-XNA
Mix #1
6 ul XNA
Mix #2
6 ul
Non-XNA
Mix #1
6 ul XNA
Mix #2
6 ul
Non-XNA
Mix #1
6 ul XNA
Mix #2
6 ul
Non-XNA
Mix #1
6 ul XNA
Mix #2
1
2
3
4
5
6
7
8
SAMPLE
1
SAMPLE
1
SAMPLE
2
SAMPLE
2
SAMPLE 3
SAMPLE 3
SAMPLE
4
SAMPLE
4
SAMPLE
5
SAMPLE
5
SAMPLE
6
SAMPLE
6
SAMPLE 7
SAMPLE 7
SAMPLE
8
SAMPLE
8
SAMPLE
9
SAMPLE
9
SAMPLE
10
SAMPLE
10
SAMPLE
11
SAMPLE
11
SAMPLE
12
SAMPLE
12
SAMPLE
13
SAMPLE
13
SAMPLE
14
SAMPLE
14
SAMPLE
15
SAMPLE
15
SAMPLE
16
SAMPLE
16
SAMPLE
17
SAMPLE
17
SAMPLE
18
SAMPLE
18
SAMPLE
19
SAMPLE
19
SAMPLE
20
SAMPLE
20
SAMPLE
21
SAMPLE
21
SAMPLE
22
SAMPLE
22
SAMPLE
23
SAMPLE
23
SAMPLE
24
SAMPLE
24
SAMPLE
25
SAMPLE
25
SAMPLE
26
SAMPLE
26
SAMPLE
27
SAMPLE
27
SAMPLE
28
SAMPLE
28
SAMPLE
29
SAMPLE
29
SAMPLE
30
SAMPLE
30
CLAMPING
CONTROL
CLAMPING
CONTROL
10 ul 2X Premix, all wells
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
FIGURE 3. S UGGESTED 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 two
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 first two wells of Row A, then 4 µl of Sample 2 into each of the next two wells in that row, and
so on, until all samples are loaded.
Pipet the clamping controls into the last two 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 or 2), a hyphen, then the sample number. For example, if running 15 samples, label tubes as S1-1,
S1-2 …..S1-15. Repeat for S2. Label the single set of Clamping Control reaction tubes as C1and C2.
When all reagents have been loaded, tightly close the PCR tubes or seal the 96-well plate to prevent
evaporation.
1.
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
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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-C2) 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 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.
TABLE 7. ACCEPTABLE CQ RANGES FOR THE CLAMPING CONTROLS
Assay
Acceptable Cq Range
Non-XNA mix #1 (C1)
23 ≤ Cq ≤ 30
BRAF 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-S2), note that the Cq values of any non-XNA mix #1
reaction should be in the range o 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
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.
Descriptions and recommendations
15
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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:
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 two reactions of each sample and all two
Clamping Control reactions, next determine ΔCq values for all the mutation sites in every
sample.
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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.
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 BRAF Mutation Detection kit were
determined by studies involving BRAF-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 BRAF gene at codon 600. These
single nucleotide polymorphisms in the BRAF 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 BRAF 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 BRAF 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
17
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
concentrations encountered in normal use impacted the ability of the QClamp BRAF Mutation
Detection Kit to distinguish between mutation-positive and mutation-negative samples.
Multiple freeze/thaw cycles
The effect of 1, 3, 5, and 8 freeze-thaw cycles were tested in QClamp BRAF Mutation Detection Kit
reagents. There is no effect up to 5 freeze-thaw cycles on the QClamp BRAF 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 BRAF 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 BRAF 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%
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%.
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
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
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
Using Roche LC96
WT without
QClamping
Profile of WT DNA Control
WT with
Qclamping
Profile of samples and controls
Understanding the Symbols
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
SYMBOLS USED IN PACKAGING
TABLE 10. SYMBOLS USED IN PACKAGING
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)
ORDERING INFORMATION:
TABLE 11. ORDERING INFORMATION
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
Product Name
Cat.Number
Size
Reader
Platform
QClamp™ Liquid Biopsy BRAF
Codon Specific Mutation
Detection Kit
DC-10-0814
30
samples
Real-time
PCR
Analysis
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BRAF Mutations
Mutations in Exon 15 codon
600
QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
TROUBLESHOOTING:
Negative result for Clamping Control with Non-XNA Mix #1
Possible Cause
Pipetting error
Inappropriate storage of kit
components
Recommended Solutions
Check pipetting scheme and setup of the reaction. Repeat the PCR
run
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
Pipetting error or omitted reagents
Inhibitory effects of the sample
material, caused by insufficient
purification
Recommended Solutions
Check pipetting scheme and the setup of the reaction. Repeat the
PCR run
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
Inappropriate storage of kit
components
Very low initial amount of target
DNA
Recommended Solutions
Store all kit components at appropriate temperature according to
label, also see Kit Components Table (Table 1)
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
PCR inhibitors present in the
reaction mixture
Inhibition by excess volume of the
RT reaction
Pipetting error or missing reagent
Annealing temperature is not
optimal
Recommended Solutions
Re-purify template DNA
Volume of the RT reaction product added to qPCR reaction should
not exceed 10% of the total qPCR reaction volume
Repeat the PCR reaction; check the concentrations of template and
primers; ensure proper storage conditions of all reagents
Optimize the annealing temperature in 3°C increments
No amplification curve but PCR product visible on a gel
Possible Cause
qPCR instrument settings are
incorrect
Inactive fluorescence detection
Instrument problems
Recommended Solutions
Check if instrument settings are correct (dye selection, reference
dye, filters)
Fluorescent detection should be activated and set at extension or
annealing/extension step of the thermal cycling protocol
Refer to the instrument manual for troubleshooting
PCR efficiency is >110%
Possible Cause
Non-specific products
Recommended Solutions
Use melting curve analysis
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
PCR efficiency is <90%
Possible Cause
PCR inhibitors present in a reaction
mixture
Recommended Solutions
Re-purify template DNA
Non-uniform fluorescence intensity
Possible Cause
Contamination of the thermal cycler
Recommended Solutions
Perform decontamination of your real-time cycler according to the
supplier’s instructions
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QClamp™ Liquid Biopsy BRAF Codon Specific Mutation Detection Kit
REFERENCES
1. Choi et al., Frequency of BRAF, BRAF, and BRAF mutations in advanced colorectal cancers:
Comparison of peptide nucleic acid-mediated PCR and direct sequencing in formalin-fixed,
paraffin-embedded tissue. Pathol Res Pract 207(12):762-8, 2011.
2. Ezzat et al., Thyroid incidentalomas. Prevalence by palpation and ultrasonography. Arch Intern
Med.154(16):1838-1840, 1994.
3. Kang et al., Prevalence, clinical and ultrasonographic characteristics of thyroid incidentalomas.
Thyroid. 14(1):29-33, 2004.
4. Kwon et al., Frequency of BRAF, BRAF, and BRAF 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.
5. Meier et al., Thyroid nodules: pathogenesis, diagnosis, and treatment. Baillieres Best Pract Res
Clin Endocrinol Metab. 14(4):559-575, 2000.
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. Rapp et al., Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus.
Proc Natl. Acad Sci USA 80(14):4218-4222, 1983.
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: information@diacarta.com
Tel: +1 510 314-8858
Fax:+1 510 735 8636
www.diacarta.com
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