QUICK REFERENCE

TaqMan® Mutation Detection Assays

Competitive Allele-Specific TaqMan® PCR
Publication Part Number 4467012 Revision Date 16 April 2012 (Rev. B)

Note: For safety and biohazard guidelines, refer to the “Safety” section in the TaqMan® Mutation Detection Assays Protocol (Part
no. 4467011). For every chemical, read the Safety Data Sheet (SDS) and follow the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves.
■ Prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
■ Perform the PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
■ Prepare the PCR mix and the PCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
■ Mutation detection experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
■ Set up the plate document or experiment and start the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■ Analyze the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Prepare the samples

Extract and purify Extract and purify gDNA samples according to your laboratory practices. We recommend the
1 gDNA gDNA extraction and purification kits listed below. For gDNA extraction from:
• FFPE tissue samples – RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE or QIAamp®
DNA FFPE Tissue Kit
• Fresh-frozen tissue samples – MELT™ Total Nucleic Acid Isolation System or Gentra
Puregene Tissue Kit
• Cell lines – PureLink® Genomic DNA Mini Kit or kit from other major supplier

Quantitate the gDNA Quantitate the gDNA by measuring the UV absorbance (A260/A280). Be sure that the human
2 gDNA that you use has an A260/A280 ratio >1.7.
Note: Given that gDNA quantitation by UV absorbance methods may not accurately reflect the
concentration of amplifiable gDNA template in some sample types (e.g. FFPE samples), also
consider using gene reference assays to assess the amount of functional template in a sample that
can be amplified by PCR.

Perform the PCR

Recommended reaction types
When setting up the reaction plate, we recommend that you include the following reaction types:

Experiment type
Reaction type
Detection ΔCT cutoff determination Mutation detection

Controls No template controls (NTC) are optional, negative NTCs are optional, negative sample controls are highly
control samples are required (inherent to the recommended, and positive control samples are
experiment), and positive controls are not necessary. recommended, if available.
 Experiment type
Reaction type
Detection ΔCT cutoff determination Mutation detection

Assays and replicates To generate accurate detection ΔCT cutoff values for a Run gDNA test samples of unknown mutation status
mutant allele assay(s), run the assay and with a mutant allele assay(s) and corresponding gene
corresponding gene reference assay on at least three reference assay. Technical replicates are not required.
wild type gDNA samples and use three technical The same amount of amplifiable DNA should be used
replicates per sample. The sample type must be the for the test sample as was used to establish the
same as the test sample. The same amount of detection ΔCT cutoff value.
amplifiable DNA should be used for each sample
tested.
In order for the Mutation Detector™ Software to compute ΔCT cutoff values or sample mutation status, you must
run each mutant allele assay, the corresponding gene reference assay, and (if applicable) technical replicates
on the same reaction plate with the same sample.
For ΔCT cutoff determination, the software can combine wild type sample ΔCT values from multiple plates to
generate an assay ΔCT cutoff value.
(Optional) TaqMan® You can duplex the IPC reagents with any TaqMan® Mutation Detection Assay to distinguish true target negatives
Mutation Detection IPC from PCR failure or inhibition.
Reagents Kit

Prepare the PCR mix and the PCR plate

Detection ΔCT cutoff determination experiments
In a detection ΔCT cutoff determination experiment, run three or more wild type gDNA samples, and three technical replicates of
each sample, with a mutant allele assay(s) and paired gene reference assay. The same amount of gDNA must be used for each
sample. Prepare the PCR mix and the PCR plate as follows:
1. For each sample, calculate the total number of reactions required.
2. Calculate the total volume required for each reaction component:
volume for 1 reaction × total no. of reactions + 10%
Note: Include 10% extra volume to compensate for pipetting errors.

Volume for 1 reaction

Component 20 µL reaction 10 µL reaction
(96-well plate) (384-well plate)

TaqMan® Genotyping Master Mix, 2X 10.0 µL 5.0 µL

Prepared gDNA sample† 2.0 – 4.0 µL 1.0 – 2.0 µL‡
(Optional) 50X Exogenous IPC Template DNA 0.4 µL 0.2 µL
(Optional) 10X Exogenous IPC Mix 2.0 µL 1.0 µL
Nuclease-free water Add water to 18.0 µL Add water to 9.0 µL
Total volume of super mix 18.0 µL 9.0 µL
Example: Total volume for 3 technical replicates (includes 10% 59.4 µL 29.7 µL
extra volume for pipetting errors)
† We recommend that you input 20 ng of gDNA; the volume of gDNA sample should not be greater than 20% of the total reaction volume.
‡ For 10-µL reactions, we recommend that you use the same amount of gDNA as for 20-µL reactions to obtain the same sensitivity of mutation detection.

3. Label a 1.5-mL microcentrifuge tube, add all components to the labeled tube, cap the tube, then vortex the tube briefly to mix the
components.

4. Centrifuge the tube briefly to spin down the contents and eliminate air bubbles.

2 TaqMan® Mutation Detection Assays Quick Reference Card

 5. For each set of technical replicates, transfer aliquots of super mix to a microcentrifuge tube, then add the TaqMan® Mutation
Detection Assay (mutant allele or gene reference assay) to each tube.

If the total reaction Add the following volumes to the microcentrifuge tube...†
volume for one TaqMan® Mutation Detection
reaction is... Super mix
Assay (10X)
20 µL 59.4 µL 6.6 µL
10 µL 29.7 µL 3.3 µL
† The super mix and assay volumes listed are for three technical replicates.

6. Add the appropriate volume of PCR mix to each reaction well of a PCR plate:

PCR plate Volume of PCR mix per reaction

96-well 20 µL
384-well 10 µL

7. Cover the plate with an optical adhesive film.

8. Centrifuge the plate briefly to spin down the contents and eliminate air bubbles.

Mutation detection experiments

In a mutation detection experiment, run the test sample with a mutant allele assay(s) and corresponding gene reference assay.
Technical replicates are not required. The amount of test sample gDNA used should be similar to the amount of wild type gDNA
sample that was used for your detection ΔCT cutoff determination experiments. Prepare the PCR mix and the PCR plate as follows:
1. For each sample, calculate the total number of reactions required.
2. Calculate the total volume required for each reaction component:
volume for 1 reaction × total no. of reactions + 10%
Note: Include 10% extra volume to compensate for pipetting errors.

Volume for 1 reaction

Component 20- µL reaction 10- µL reaction
(96-well plate) (384-well plate)
TaqMan® Genotyping Master Mix, 2X 10.0 µL 5.0 µL
Prepared gDNA sample† 2.0 – 4.0 µL 1.0 – 2.0 µL‡
(Optional) 50X Exogenous IPC Template DNA 0.4 µL 0.2 µL
(Optional) 10X Exogenous IPC Mix 2.0 µL 1.0 µL
Nuclease-free water Add water to 18.0 µL Add water to 9.0 µL
Total volume of super mix 18.0 µL 9.0 µL
† We recommend that you input 20 ng of gDNA; the volume of gDNA sample should not be greater than 20% of the total reaction volume.
‡ For 10-µL reactions, we recommend that you use the same amount of gDNA as for 20-µL reactions to obtain the same sensitivity of mutation detection.

3. Label a 1.5-mL microcentrifuge tube, add all components to the labeled tube, cap the tube, then vortex the tube briefly to mix the
components.

4. Centrifuge the tube briefly to spin down the contents and eliminate air bubbles.
5. Add the appropriate volume of super mix to each reaction well of a PCR plate:

PCR plate Volume of super mix per reaction

96-well 18 µL
384-well 9 µL

TaqMan® Mutation Detection Assays Quick Reference Card 3

 6. Add the appropriate volume of TaqMan® Mutation Detection Assay
(mutant allele or gene reference assay) to each reaction well:

Volume of TaqMan® Mutation Detection Assay

PCR plate
(10X) per reaction

96-well 2 µL
384-well 1 µL

7. Cover the plate with an optical adhesive film.

8. Centrifuge the plate briefly to spin down the contents and eliminate air bubbles.

Set up the plate document or experiment and start the run

In the real-time PCR system software:
1. Select the experiment type: Absolute Quantitation or Quantitation - Standard Curve.
2. For each well that contains a reaction, apply a sample name, assay name, and target or detector name. For downstream analysis
with the Mutation Detector™ Software, note the following:

Parameter Comments
Sample Name If you enter the sample name as NTC, the Mutation Detector™ Software treats the sample as
a No Template Control.
Apply the same sample name to all wells containing the sample and mutation detection assays
that will be analyzed together. If the sample name is not identical for each well across the
reaction plate, the Mutation Detector Software treats these as different samples.
If you are using technical replicates, apply the same sample name to the wells of each technical
replicate group.
The Mutation Detector™ Software combines data from replicate wells only if the wells share the
same sample name. If the replicate wells are named differently, the software analyzes the wells
as different samples.
Target Name or Detector In order for the Mutation Detector™ Software to correctly analyze the data, you must use Applied
Name Biosystems assay names. Using Applied Biosystems assays names ensures that a mutant allele
assay will be paired with the appropriate gene reference assay in the analysis.
If the IPC reagents are duplexed in the reaction, enter IPC as the detector name.
Reporter and Quencher For wells that contain:
Names • TaqMan® Mutation Detection Assays, FAM™ dye is the reporter and Non Fluorescent or NFQ-
MGB is the quencher
• IPC reagents (from the TaqMan® Mutation Detection IPC Reagent Kit), VIC® dye is the
reporter and TAMRA™ dye is the quencher
Note: Instrument results files exported from the 7500 SDS v1.X software do not contain a
Reporter column with dye names. In this case, the Mutation Detector™ Software uses the assay
name (from the Detector/Target column) to determine the dye name. If the assay name is IPC,
the software assumes that the reporter is VIC® dye; for all other assay names, the software
assumes that the reporter is FAM™ dye.

3. Enter sample quantity values in the real-time PCR system software:

a. Select Standard as the task for each well of interest.
b. Enter a numeric value. We recommend that the numeric values you enter are relevant to the ng amount of gDNA or copies of
DNA input.

4 TaqMan® Mutation Detection Assays Quick Reference Card

 For downstream analysis with the Mutation Detector™ Software, note the following:

Parameter Comments

Amount of sample DNA For all mutation detection assay results that will be used to calculate a detection ΔCT cutoff
value or mutation status, load the same amount of sample DNA into the wells. If the sample
quantity is not specified in the real-time PCR system software, then the Mutation Detector™
Software assumes that the sample amounts in each well are equivalent.
Sample quantity value The sample quantity value must be equal and must be provided in the real-time PCR system
software for all samples that will be analyzed together if you are calculating calibration ΔCT
values in real-time (comparing positive control sample CT values between a mutant allele assay
and a corresponding gene reference assay).

4. Set the following thermal-cycling conditions:

• Run mode – Standard
• Sample volume – 10 µL (384-well plates) or 20 µL (96-well plates)
• Thermal-cycling profile – See the table below

Time
Stage Temp. Cycles Data collection
(mm:ss)
1 95°C 10:00 1 No
2 92°C 00:15 5 No
58°C 01:00 No
3 92°C 00:15 40 No
60°C 01:00 FAM™ or VIC® dye†
† FAM dye is the reporter for TaqMan Mutation Detection Assays; VIC dye is the reporter for the IPC
reagents (from the TaqMan Mutation Detection IPC Reagent Kit).

5. Load the reaction plate into the real-time PCR instrument, then start the run.

Analyze the data

For detailed analysis procedures, refer to the Mutation Detector™ Software User Guide (Part no. 4467102). Briefly, the analysis steps are:
1. Analyze the data in the real-time PCR system software, using the following analysis settings:
• Manual CT (threshold cycle): 0.2
• Automatic Baseline: On
The real-time PCR system software determines the CT values for the mutation detection assays and (optional) IPC reagent
reactions.

2. View the amplification plots and/or CT values for all reaction wells as follows:

Reaction type What to look for

Samples tested with gene Verify that the amplification curves have a distinct, linear amplification phase and that the
reference assays (FAM™ dye CT values are within a range of ~18 to 28 for a 20-µL reaction and ~17 to 27 for a 10-µL
signal) reaction.
Samples tested with mutant allele Review the amplification curves and CT values.
assays (FAM™ dye signal)
The presence or absence of a distinct, linear amplification phase and CT values depend on
the amount of mutant allele present in the sample.
Positive control samples Verify that the amplification curves have a distinct, linear amplification phase and that the
CT values are within the expected range for the quantity of target present in the sample.
Note: Positive control samples that are used to calculate calibration ΔCT values must
contain 100% target sequence for the mutation detection assay that is run with the positive
control.
Negative control samples Verify that the negative control samples either do not amplify or have very high CT values.

TaqMan® Mutation Detection Assays Quick Reference Card 5

 Reaction type What to look for
No template control (NTC) Verify that the NTC samples do not amplify.
samples
Technical replicates Verify that the CT values are similar between replicates.
Note: Some variance is expected between replicates for samples that contain low
amounts of target allele and have high CT values.
(If using) IPC reagents, from the Verify that the amplification curves (VIC® dye signal) in all samples have a distinct, linear
TaqMan® Mutation Detection IPC amplification phase and that the CT values are similar for all wells that contain the same
Reagent Kit sample

Note: Some mutant allele assays are expected to give low level non-specific amplification of wild type gDNA samples. The
TaqMan® Mutation Detection Assay Index file (download from: www.lifetechnologies.com/castpcr) provides off-target
amplification CT values determined for each mutant allele assay that can be used to evaluate an assay's performance. Note that
the off-target CT value may differ for different sample types.

3. Export the Results or Results Table from the real-time PCR system software as a *.csv or *.txt file.
4. Import the *.csv or *.txt file(s) into the Mutation Detector™ Software. The Mutation Detector Software can:
• Calculate detection ΔCT cutoff values
• Determine the presence or absence of a mutation in a sample and
• Quantitate the percent mutation in a sample (when assay calibration values are available)

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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