PRODUCT BULLETIN KRAS Mutation Analysis Reagents BRAF Mutation Analysis Reagents Keep pace with current oncology research The Applied Biosystems ® KRAS Mutation Analysis Reagents and BRAF Mutation Analysis Reagents were developed to facilitate research aimed at elucidating the role of these regulatory proteins in oncology. Offering an off-the-shelf solution, the KRAS and BRAF reagent sets provide a straightforward protocol with results analyzed on the industry-standard Applied Biosystems ® 3500 Series or 3130 Series Genetic Analyzers (see sidebar on Applied Biosystems ® Genetic Analyzers). This allows you to easily integrate KRAS and BRAF mutation analysis into your laboratory’s Growth factors, e.g. EGF EGFR Kinase Kinase Cell proliferation Cell membrane Figure 1. The protein products of the KRAS and BRAF genes perform essential functions in the epidermal growth factor (EGF) signaling pathway. Because of its involvement in the regulation of cell proliferation, the EGF pathway is studied by many cancer researchers. capabilities and to maximize the utility of your Applied Biosystems ® genetic analysis instrument—well established for excellence in sequencing and fragment analysis applications. Simple, streamlined workflow for accurate KRAS and BRAF mutation analysis The protocol for the KRAS and BRAF Mutation Analysis Reagents is simple and efficient (Figure 2). The KRAS reagent set amplifies 12 important SNPs in two multiplex PCR analyses: 6 SNPs for codon 12 in a single multiplex, and 6 SNPs for codon 13 in an additional multiplex. Similarly, the BRAF Figure 2. Experimental workflow for identifying KRAS and/or BRAF variants. Extracted DNA KRAS: 12 mutations, 2 tubes BRAF: 3 mutations, 1 tube The epidermal growth factor receptor (EGFR) pathway is a complex signaling cascade that is associated with the development and progression of many cancer conditions (Figure 1). KRAS and BRAF gene mutations are present in a number of cancers, including those of the colon, lung, pancreas, biliary tract, endometrium, and ovary. It has been shown that approximately 35% to 45% of metastatic colorectal cancer (mCRC) tumors may have a KRAS or BRAF mutation [1–3], which makes them less likely to respond to anti-EGFR therapies. Identifying these mutations is therefore of great importance in clinical and pharmaceutical research. KRAS Mutation Analysis Reagents BRAF Mutation Analysis Reagents • Simple analysis—unambiguous, easy- to-interpret results • Thorough coverage—detects 12 mutations in the KRAS gene and 3 in the BRAF gene • Sensitive—able to detect 1–5% mutation contribution in a background of wild type genomic DNA from analytical samples • Efficient—interrogate multiple loci from the same sample in a minimum number of tubes
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Keep pace with current oncology researchThe Applied Biosystems® KRAS Mutation Analysis Reagents and BRAF Mutation Analysis Reagents were developed to facilitate research aimed at elucidating the role of these regulatory proteins in oncology. Offering an off-the-shelf solution, the KRAS and BRAF reagent sets provide a straightforward protocol with results analyzed on the industry-standard Applied Biosystems® 3500 Series or 3130 Series Genetic Analyzers (see sidebar on Applied Biosystems® Genetic Analyzers). This allows you to easily integrate KRAS and BRAF mutation analysis into your laboratory’s
Growth factors,e.g. EGF
EGFR
KinaseKinase
Cellproliferation
Cell membrane
Figure 1. The protein products of the KRAS and BRAF genes perform essential functions in the epidermal growth factor (EGF) signaling pathway. Because of its involvement in the regulation of cell proliferation, the EGF pathway is studied by many cancer researchers.
capabilities and to maximize the utility of your Applied Biosystems® genetic analysis instrument—well established for excellence in sequencing and fragment analysis applications.
Simple, streamlined workflow for accurate KRAS and BRAF mutation analysisThe protocol for the KRAS and BRAF Mutation Analysis Reagents is simple and efficient (Figure 2). The KRAS reagent set amplifies 12 important SNPs in two multiplex PCR analyses: 6 SNPs for codon 12 in a single multiplex, and 6 SNPs for codon 13 in an additional multiplex. Similarly, the BRAF
Figure 2. Experimental workflow for identifying KRAS and/or BRAF variants.
Extracted DNA
KRAS: 12 mutations, 2 tubes
BRAF: 3 mutations, 1 tube
The epidermal growth factor receptor (EGFR) pathway is a complex signaling cascade that is associated with the development and progression of many cancer conditions (Figure 1). KRAS and BRAF gene mutations are present in a number of cancers, including those of the colon, lung, pancreas, biliary tract, endometrium, and ovary. It has been shown that approximately 35% to 45% of metastatic colorectal cancer (mCRC) tumors may have a KRAS or BRAF mutation [1–3], which makes them less likely to respond to anti-EGFR therapies. Identifying these mutations is therefore of great importance in clinical and pharmaceutical research.
• Thorough coverage—detects 12 mutations in the KRAS gene and 3 in the BRAF gene
• Sensitive—able to detect 1–5% mutation contribution in a background of wild type genomic DNA from analytical samples
• Efficient—interrogate multiple loci from the same sample in a minimum number of tubes
Applied Biosystems® 3130 Series** and 3500 Series Genetic AnalyzersApplied Biosystems capillary electrophoresis platforms are the industry standard, providing the most reliable, efficient, and widely published technology for DNA sequencing and fragment analysis. Because of its high resolution, throughput capabilities, ease of use, and small sample requirements, capillary electrophoresis (CE) using Applied Biosystems® Genetic Analyzers is the pre-ferred method for numerous DNA analysis applications. Using Applied Biosystems® Genetic Analyzers, single-base resolution can be obtained on DNA fragments of up to several hundred base pairs, an important consideration for researchers investigating KRAS and BRAF mutations.
Applied Biosystems offers a number of CE-based genetic analyzers (see table below). For state-of-the-art, intelligent hardware, intuitive software, and innovative consumables, select the 8-capillary 3500 or 24-capillary 3500xL system. From single-capillary to large, production-scale systems, Applied Biosystems® Genetic Analyzers are designed to support your critical life science research needs.
reagent set analyzes 3 important SNPs in a single multiplex PCR (Table 1). Both reagent sets are optimized using the same PCR thermal cycling conditions and the same run modules for capillary electrophoresis on either the 3500 Series or the 3130 Series Genetic Analyzers. This allows you to analyze samples amplified by either the KRAS or BRAF reagent sets concurrently and reliably.
Shifted termination assay (STA) yields unambiguous resultsThe KRAS and BRAF Mutation Analysis Reagents employ a proprietary shifted termination assay (STA) technology to amplify the mutation signal. The assay is performed using specially designed primers, enzyme master mix, and chemistry protocol. The STA reaction recognizes wild type or mutant target sequences and selectively extends the detection primer with 1 to 20 nucleotides to generate various lengths of primer extension products. The extended STA fragments are separated by capillary electrophoresis with an Applied Biosystems® 3500 Series or 3130 Series Genetic Analyzer, followed by data analysis using GeneMapper® Software.
Table 1. Mutations Detected Using the KRAS and BRAF Mutation Analysis Reagents.
Mutations Interrogated Using the KRAS Mutation Analysis Reagents
Mutations Interrogated Using the BRAF Mutation Analysis Reagents
*The capillary array length is the well-to-read length. **Available as factory-refurbished instruments. †Sample capacity is the number of samples or plate types the autosampler can accommodate.
Figure 5. Using GeneMapper® Software v4.1, KRAS and BRAF mutations are clearly observed. With a single exception (BRAF GTG>GGG mutation), the peak corresponding to the mutation always appears to the left of the peak corresponding to the wild type.
GeneMapper® Software v4.1 offers easy data analysisGeneMapper® Software is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems (Figure 4).GeneMapper® Software v4.1 offers a simple, qualitative method for interpreting fragment analysis data, where anyone can easily identify mutations present in the sample (Figure 5). To streamline the data analysis, a bin setup protocol for assistance in KRAS and BRAF analysis using GeneMapper® Software v4.1 is provided in the Documents: Manuals & Protocols section for these products at lifetechnologies.com/KRASanalysis and lifetechnologies.com/BRAFanalysis.
References1. Di Nicolantonio F, Martini M, Molinari F et
al. (2008) Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 26(35):5705–5712.
2. Karapetis CS, Khambata-Ford S, Jonker DJ et al. (2008) K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359(17):1757–1765.
3. Goncalves A, Esteyries S, Taylor-Smedra B et al. (2008) A polymorphism of EGFR extracellular domain is associated with progression free-survival in metastatic colorectal cancer patients receiving cetuximab-based treatment. BMC Cancer 8:169.
Figure 4. GeneMapper® Software v4.1 offers a powerful, streamlined data interface. Using GeneMapper® Software v4.1, data review options are flexible and intuitive and results are clearly presented, making KRAS and BRAF mutation analysis very clear.
Select KRAS from the Plot Setting pull-down menu
Select the number of panes to display
Click to show and hide dye colors
Click to show combined or separate dyes
Click to display No table, Samples table, or Genotypes table
Click, Ctrl-click or Shift-click to select peaks and highlight the corresponding rows of data in the table below
For additional information on the KRAS and BRAF Mutation Analysis Reagents and to place your order, go to lifetechnologies.com/KRAS or lifetechnologies.com/BRAF
A Scenario for Using Both the KRAS and BRAF Mutation Analysis Reagents in a Common Research Situation
In certain research scenarios, analysis of these pathways may include a stepwise decision in which the reagents for identifying KRAS variants are used first. If KRAS variants are present, mutation is reported and no further investigation is performed. If no KRAS variants are found, the same samples are then screened for BRAF variants. Similarly, if variants are present, the observed mutation(s) is reported. For labs that need a fast result, both gene regions can be investigated simultaneously.
Mutation
No Mutation
Sample KRASDetection
BRAFDetection
Report
Report
Analysis
Analysis
Ordering information
Product Analysis Reagents Components Number of Samples Analyzed