PRODUCT BULLETIN KRAS Mutation Analysis Reagents BRAF Mutation Analysis Reagents

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.

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

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

KRAS Codon 12 Mutations KRAS Codon 13 Mutations BRAF Mutations

Gly12Ser (GGT>AGT) Gly13Ser (GGC>AGC) Val600Glu (GTG>GAG)

Gly12Arg (GGT>CGT) Gly13Arg (GGC>CGC) Val600Gly (GTG>GCG)

Gly12Cys (GGT>TGT) Gly13Cys (GGC>TGC) Val600Ala (GTG>GGG)

Gly12Asp (GGT>GAT) Gly13Asp (GGC>GAC)

Gly12Ala (GGT>GCT) Gly13Ala (GGC>GCC)

Gly12Val (GGT>GTT) Gly13Val (GGC>GTC)

Figure 3. KRAS and BRAF Mutation Analysis Reagent Sets.

Applied Biosystems® Genetic Analyzers for the KRAS and BRAF Mutation Analysis Workflow.

Instrument Number of Capillaries Capillary Array Length* Polymer Type Sample Capacity†

3500 Genetic Analyzer 8 36, 50 POP-7™, POP-4™, POP-6™ 96- and 384-well plates;96-well Fast and 8-tube standard or Fast strips

3500xL Genetic Analyzer

24 36, 50 POP-7™, POP-6™ 96- and 384-well plates;96-well Fast and 8-tube standard or Fast strips

3130 Genetic Analyzer 4 36, 50, 80 POP-7™, POP-4™, POP-6™ 96- and 384-well plates

3130xl Genetic Analyzer 16 36, 50, 80 POP-7™, POP-4™, POP-6™ 96- and 384-well plates

*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

Zoom by click-dragging on x or y axis

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4,80027 29 31 33 35 37 39 41

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35 37 39 41

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1,600

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49 53 57 61 65

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47 51 55 6359

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4,00043 49 55 61

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800

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45 49 53 57 61 65

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55 57 59 61 63

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47 51 55 59 63

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6,000

44 46 48 50 52 54

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44 46 50 5448 52

44 46 50 5448 52

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4,00047 49 51 53 55

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43 45 5147 555349 57

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6,00047 49 51 53

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Wild TypeWild TypeWild Type

Wild Type Wild Type

Wild TypeWild Type

GGC>GACGGT>GTT

GGT>TGT

GGT>GCT

GGT>CGT

GGT>GAT

GGT>AGT

GGC>CGC

GGC>TGC

GGC>AGC

GGC>GTC

GGC>GTC

GTG>GCG

GTG>GGG

GTG>GAGWild Type

Wild Type

Wild Type

Wild Type

Wild Type Wild Type

Wild TypeWild Type

Codon 12 Codon 13 Codon 600

KRAS BRAF

For Research Use Only. Not for use in diagnostic procedures. ©2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. CO02083 1212

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

KRAS Mutation Analysis Reagents DNA Amplification Master Mix, 1,000 μL 30 samples KRAS PCR Primers, 50 μLKRAS Detection Primers, Codon 12, 80 μLKRAS Detection Primers, Codon 13, 80 μLClean-Up Enzyme Mix, 430 μLKRAS Controls, Codon 12, 25 μLKRAS Controls, Codon 13, 25 μLKRAS Enrichment Mix, Codon 12, 430 μLKRAS Enrichment Mix, Codon 13, 430 μLLoading Buffer (two tubes), 1,200 μL each tubeKRAS TF-50 Filter TipsCollection Tubes

BRAF Mutation Analysis Reagents DNA Amplification Master Mix, 1,000 μL 30 samplesBRAF PCR Primers, 50 μLBRAF Detection Primers, 80 μLClean-Up Enzyme Mix, 430 μLBRAF Control Mix, 50 μLBRAF Enrichment Mix, 430 μLLoading Buffer, 1,200 μLBRAF TF-50 Filter TipsCollection Tubes