qBiomarker Somatic Mutation PCR Assays

For detecting the presence of specific DNA sequence mutations in cancer and oncogenesis

Features

  • Simple real-time PCR procedure
  • High sensitivity and wide dynamic range
  • Designed for routine use on most qPCR instruments
  • Master mix is included
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qBiomarker Somatic Mutation PCR Assays

Cat. No. / ID: 337011

PCR assay and master mix
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qBiomarker Somatic Mutation PCR Assays
qBiomarker Mutation Assay Control DNA
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qBiomarker Somatic Mutation PCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

qBiomarker Somatic Mutation PCR Assays identify the presence of individual specific sequence mutations present in cell lines or research samples that are critical for toxicological, drug development, and cancer studies. The mutations are selected from comprehensive curated databases (e.g., COSMIC) and literature reviews based on their clinical or functional relevance and frequency in patient populations.

Principle

Real-time PCR is the most sensitive and reliable method for the detection of DNA mutations. By combining allele-specific amplification and hydrolysis probe detection, qBiomarker Somatic Mutation real-time PCR assays have been developed which can detect as few as 1% somatic mutations in the background of wild-type genomic DNA. Allele-specific amplification is achieved by Amplification Refractory Mutation System (ARMS) technology, which is based on the discrimination by Taq polymerase between a match and a mismatch at the 3’ end of the PCR primer (see figure " Principle of ARMS technology").
See figures

Procedure

After the isolation of genomic DNA from fresh, frozen, or fixed samples, add an aliquot of each sample to a separate real-time PCR tube containing the appropriate master mix (included) and the qBiomarker Somatic Mutation PCR Assay. Then, add an aliquot of each sample to a separate real-time PCR tube containing the appropriate master mix (included) and the corresponding reference gene copy assay. Finally, run the recommended cycling program.

Determine the CT values for each mutation-specific assay and the corresponding reference gene copy assay for each sample using your instrument's software. Then, paste the values into the correct Excel-based data analysis template to determine which samples contain the tested mutation.

Applications

qBiomarker Somatic Mutation PCR Assays are highly suited for the rapid and accurate identification of individual specific sequence mutations present in fresh, frozen, or fixed samples.

Supporting data and figures

Resources

Instrument Technical Documents (2)
For gene expression and genomic analysis
For screening disease-focused mutation panels by PCR
Kit Handbooks (1)
For real-time PCR-based, pathway- or disease-focused somatic mutation profiling

FAQ

How do I use the qBiomarker Somatic Mutation PCR Arrays?
The simple workflow involves mixing the DNA sample of interest with ready-to-use qBiomarker Probe Master Mix, aliquoting the mixture into the array plate wells, performing real-time PCR, and making mutation/genotype calls using web-based data analysis software or Excel-based templates.
FAQ ID -2923
What PCR machine do I need to use to run such a qBiomarker Somatic Mutation PCR Arrays?

The arrays are designed for routine use on any PCR instruments. Arrays are available in the following formats:

qBiomarker Somatic Mutation PCR Array Format A: Fluoroscein, 96-well; for Bio-Rad iCycler, iQ5, MyiQ, and MyiQ2 instruments

qBiomarker Somatic Mutation PCR Array Format A: ROX, 96-well; for ABI Standard 96-well Blocks (5700, 7000, 7300, 7500, 7900HT, ViiA 7); Bio-Rad Chromo 4 (MJ Research); Stratagene Mx3005p, Mx3000p; Eppendorf ep realplex 2/2S, and 4/4S instruments

qBiomarker Somatic Mutation PCR Array Format C: ROX, 96-well; for ABI 7500 FAST 96-well Block, 7900HT FAST 96-Well Block, StepOnePlus, and ViiA 7 FAST 96-well Block instruments

qBiomarker Somatic Mutation PCR Array Format D: ROX, 96-well; for Bio-Rad CFX96, Opticon and Opticon 2 (MJ Research); Stratagene Mx4000 instruments

qBiomarker Somatic Mutation PCR Array Format E: ROX, 384-well; for ABI 7900HT 384-well Block, ViiA 7 384-well Block; Bio-Rad CFX384 instruments

qBiomarker Somatic Mutation PCR Array Format F: ROX, 96-well; for Roche LightCycler 480 96-well Block instruments

qBiomarker Somatic Mutation PCR Array Format G : ROX, 384-well; for Roche LightCycler 480 96-well Block instruments

qBiomarker Somatic Mutation PCR Array Format R : ROX, 100-well disc; for QIAGEN Rotor-Gene Q/6000 instruments

FAQ ID -2917
What sample types can I test on the qBiomarker Somatic Mutation PCR Arrays?
The somatic mutation detection assays and arrays prove to yield accurate and verifiable results in various sample types, including fresh frozen cell lines and tissue samples, cell line admixtures, FFPE cell line samples and FFPE tissue samples from various sources.
FAQ ID -2919
How much DNA starting sample is needed for analysis with a Somatic Mutations PCR Array? And Assay?
The recommended starting DNA sample for the Somatic Mutation PCR Arrays is 500 ng for a 96-well plate, and 200 ng for a 384-well plate. If using less, please proceed with Whole Genome Amplification (see User Manual for protocol); if using DNA isolated from FFPE samples, 500 ng – 2 ug is recommended for 96-well plates & 200 ng – 1.6 ug is recommended for 384-well plates. For Somatic Mutation Assays, it is recommend to start with 5 – 40 ng.
FAQ ID -2431
How are the different mutations for a single gene normalized on the qBiomarker Somatic Mutation PCR array?
Each qBiomarker mutation assay has it corresponding gene assay on the array, in the form of a Copy Number Assay.
FAQ ID -2428
How can I check DNA sample quality before using the Somatic Mutation PCR Arrays & Assays?
We recommend using the QIAGEN QIAamp DNA Mini Kit for DNA isolation, with the recommended RNase step. For sample quality, we recommend assaying for DNA concentration & purity with a UV Spectrophotometer; for DNA Integrity, an agarose gel should be run; and DNA quality and consistency can be checked on the Somatic Mutations QC plate that measures 7 reference genes by RT-PCR.
FAQ ID -2430
How do you decide on the qBiomarker Somatic Mutation PCR Arrays to include on the pathway arrays and disease arrays?
For pathway-focused arrays, we included assays for detecting the most frequent and functionally verified mutations for multiple genes within a specific pathway implicated in a variety of cancers. Additional assays are also available for each gene to allow array customization. For disease-focused arrays, we selected top somatic mutations for that disease type covering between 4,000 and 40,000 published tumor samples for each disease type.
FAQ ID -2913
What pathways and diseases are currently covered for the qBiomarker Somatic Mutation PCR Arrays?
The pathways covered include major receptor tyrosine kinase pathways, non-receptor kinase pathways, as well as additional oncogene and tumor suppressor pathways; and the targeted diseases include all major cancer types. In addition, a collection of more than 800 pre-validated somatic mutation assays enables researchers to study single mutations or to customize the mutation panels or collections according to their research needs.
FAQ ID -2922
What are the technological principles behind somatic mutation PCR arrays and assays?
Real-time PCR is the most sensitive and reliable method for the detection of DNA mutations. By combining allele specific amplification and hydrolysis probe detection, we have developed real-time PCR assays that detects as low as 1% somatic mutations in the background of wild-type genomic DNA. Allele specific amplification is achieved by Amplification Refractory Mutation System (ARMS@) technology, which is based on the ability of Taq polymerase to discriminate between a match and a mismatch at the 3' end of the PCR primer.
FAQ ID -2924
How many mutations can I profile per sample on the qBiomarker Somatic Mutation PCR Array?
Depending on the qBiomarker Somatic Mutation PCR Array that is chosen, one can profile between 40 and 360 mutations per sample in one PCR run.
FAQ ID -2911
What method(s) do you recommend as an alternative way to validate the mutations identified on the somatic mutation PCR arrays?
Sanger sequencing and pyrosequencing can be used to validate the mutations identified on the arrays. However, one needs to bear in mind that the detection sensitivity of Sanger sequencing is around 20%, and the detection sensitivity of pyrosequencing is around 5%, while RT-PCR based arrays can detect 1% or lower mutations. Therefore, mutations occurring below the detection limit of Sanger sequencing and pyrosequencing will not be verified by these two methods.
FAQ ID -2920
Where are the somatic mutations published?
The content for the Somatic Mutations PCR Array is derived from the COSMIC database, established by the Sanger Institute of the Wellcome Trust. (http://www.sanger.ac.uk/genetics/CGP/cosmic/)
FAQ ID -2429
What is the principle for qBiomarker Somatic Mutation PCR Array data analysis?
The basic principle behind the data analysis is that we compare the Ct value of a mutation assay in a test sample with the Ct value of the same assay in a wildtype sample. When there is a significant difference (a preset value of 4 Cts) between the Ct values, the test sample is concluded to contain the mutation. The Ct values used for comparison can either be raw Ct (in average Ct method) or normalized Ct (in delta delta Ct case). When the Ct difference falls between 3 and 4, we give a borderline mutation call, which means that the mutation may be present at low percentage. When the Ct difference is smaller than 3, we give a negative mutation call (i.e. the mutation percentage is beyond the detection limit of the array). The wildtype sample can be either a genuine wildtype sample that is tested in the same experiment, or it could be a "virtual" wildtype sample that is computed from all test samples. For detailed description of the data analysis principle, refer to (link to white paper).
FAQ ID -2916
What are the control features on the qBiomarker Somatic Mutation PCR Arrays?
Each array contains gene copy reference assays for each gene represented by the array. These assays target non-variable regions of the genes and measure input DNA quality and amount. In addition, these assays sensitively measure gene dosage to normalize mutation assay data against the gene copy number. Each array also contains positive PCR controls (SMPC) to test for the presence of inhibitors in the sample or the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it.
FAQ ID -2914
What are the advantages of RT-PCR based somatic mutation PCR arrays and assays compared to other platforms?
The main advantages are qPCR-based superior detection sensitivity and straightforward data analysis procedure. Additional major advantages over other currently available mutation detection platforms/methods are: (1) The workflow is very simple, involving only one setup step. No multi-step handling is involved, and hands-on time is less than any other method available. (2) Reactions involved are all closed-tube reactions avoiding sample contamination. (3) The DNA sample input is low. (4) The hardware involved in analysis using the mutation detection arrays and assays is highly accessible, enabling such analysis for any laboratory with access to real-time PCR instruments.
FAQ ID -2921
What is the sensitivity of the assays contained on the qBiomarker Somatic Mutation PCR Arrays?
The assays were validated to have at least 1% sensitivity (i.e. able to detect 1% mutation on a wildtype background), but the sensitivity is usually higher. On average, the assay sensitivity is 0.03%.
FAQ ID -2910
How many different genes and gene mutations are on a Somatic Mutation PCR Array?
There are currently 13 different pathway-focused and 10 disease-focused Somatic Mutation PCR Arrays available, with each array assaying from 3 to 19 genes, and on average 5-30 mutations within each gene. Please refer to the individual product sheets/ product webpage for a full listing.
FAQ ID -2427
What effects can be predicted with poor sample quality? How robust is the platform with questionable sample quality (such as FFPE samples)?

Poor quality samples tend to give higher Cts in all assays (mutation assays and gene copy number assays) and there are two possible consequences:

 (1) if using average Ct method for data analysis, even real mutations in poor quality samples will not be called, because the mutation locus Ct will be pushed to a high Ct region; (2) if using delta delta Ct method for data analysis, there will be a number of false positives in low quality samples.

We recommend using the average Ct value for gene copy number assays on the array to gauge the sample quality (or run the sample on a DNA QC plate before running samples on an array). For FFPE samples, we recommend the average Ct to be below 32 to allow sensitive detection of mutations. Samples that meet this criterion perform robustly on the arrays.

FAQ ID -2918
What is the data analysis method, and where can I find it for the qBiomarker Somatic Mutatation PCR arrays?

The choice between one of two data analysis methods depends on the experimental setup and sample type.

1. ΔΔCt Method Recommended for experiments using: Small (four or less) number of fresh, frozen samples Large number of samples with similar DNA quality

2. Average Ct Method Recommended for experiments using: FPPE samples, large number of samples or without wild-type control samples

Data analysis can either be performed on the somatic mutation data analysis web portal or by downloading the Excel data analysis templates at http://www.sabiosciences.com/somaticmutationdataanalysis.php

FAQ ID -2915
Were all assays on the qBiomarker Somatic Mutation PCR Arrays bench-validated?
Each array contains a panel of assays bench-validated for hydrolysis probe based real-time RT-PCR detection. These assays are optimized to work under standard cycling conditions enabling a large number of assays to be analyzed simultaneously.
FAQ ID -2912