For in vitro diagnostic use. Others cobas 4800 BRAF V600 Mutation Test IVD cobas® 4800 BRAF V600 Mutation Test RMD-4800-BRAF-002 05 985 595 190 5 985 595 190 05985595190 5985595190 05985595190 KIT c4800 BRAF v600E AMP/DET 24T CE-IVD cobas 4800 BRAF V600 Mutation Test 00875197004144 Reagents, kits 1 kit 24 tests false The cobas® 4800 BRAF V600 Mutation Test is an in vitro diagnostic device intended for the qualitative detection of BRAF V600E mutation in DNA extracted from formalin-fixed, paraffin-embedded human melanoma tissue. The cobas® 4800 BRAF V600 Mutation Test is a real-time PCR test on the cobas® 4800 System, and is intended to be used as an aid in selecting melanoma patients for treatment with the targeted therapies listed in the table below, in accordance with the approved therapeutic product labeling:TherapeuticTherapeutic IndicationTest ResultZELBORAF® (vemurafenib)BRAF V600EMutation DetectedCOTELLIC® (cobimetinib), in combination with ZELBORAF® (vemurafenib)BRAF V600E or V600K*Mutation Detected**Due to cross-reactivity by the cobas® 4800 BRAF V600 Mutation Test, the clinical trial for cobimetinib, in combination with vemurafenib, included some patients whose tumor carried the BRAF V600K mutation. en The cobas® BRAF V600 Mutation Test is an in vitro diagnostic device intended for the qualitative detection of BRAF V600E mutation in DNA extracted from formalin-fixed, paraffin-embedded human melanoma tissue. The cobas® BRAF V600 Mutation Test is a real-time PCR test on the cobas® 4800 System, and is intended to be used to identify patients with melanoma whose tumors harbor the V600E mutation of BRAF. en The primary use of the cobas® 4800 BRAF V600 Mutation Test is the detection of the BRAF V600 mutations in DNA extracted from formalin-fixed, paraffin-embedded human melanoma and papillary thyroid carcinoma (PTC) tissue. In melanoma, it is intended to be used as an aid in selecting patients whose tumors carry BRAF V600 mutations for treatment either with ZELBORAF® (vemurafenib) alone, or with COTELLIC® (cobimetinib) in combination with ZELBORAF® (vemurafenib). en The cobas® 4800 BRAF V600 Mutation Test ( cobas BRAF Test) is based on two processes: (1) manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET); (2) PCR amplification and detection target DNA using a complementary primer pair and two oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wild-type BRAF V600 sequence and one is designed to detect the V600E mutation sequence. Two external run controls are provided and the wild-type allele serves as an internal, full process control.Specimen PreparationFFPET specimens are processed and genomic DNA isolated using the cobas® DNA Sample Preparation Kit, a manual specimen preparation based on nucleic acid binding to glass fibers. A deparaffinized 5-μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration to be added to the amplification and detection mixture. The target DNA is then amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas BRAF Test kit.PCR Amplification and DetectionTarget SelectionThe cobas BRAF Test uses primers that define a 116-base pair sequence of human genomic DNA containing the BRAF codon 600 site in exon 15. The entire BRAF gene is not amplified. The cobas BRAF Test is designed to detect the nucleotide (T1799A) change in the BRAF gene which results in a valine-to-glutamic acid substitution at codon 600 (V600E). BRAF wild-type and mutant DNA target-specific, fluorescent dye-labeled TaqMan probes bind to the wild-type and mutant sequences, respectively. The wild-type and mutant sequences are detected using a dedicated optical channel for each sequence.Target AmplificationThermus species Z05 DNA polymerase is utilized for target amplification. First, the PCR reaction mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA Polymerase, in the presence of divalent metal ion and excess dNTPs, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy of the targeted 116-basepair region of the BRAF gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA. Amplification occurs only in the region of the BRAF gene between the primers.Automated Real-time DetectionThe cobas BRAF Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Two different reporter dyes are used to label the target-specific BRAF wild-type (WT) probe and the BRAF V600E mutation probe. Amplification of the two BRAF sequences can be detected independently in a single reaction well by measuring fluorescence at the two characteristic wavelengths in dedicated optical channels.Selective AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas BRAF Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).11 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon due to the use of dUTP as one of the nucleotide triphosphates in the Reaction Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Reaction Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. en The cobas BRAF Test is based on two major processes: (1) manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET); (2) PCR amplification of target DNA using a complementary primer pair and two oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wild-type BRAF V600 sequence and one is designed to detect the V600E mutation sequence. Two external run controls are provided and the wild-type allele serves as an internal, full process control.Specimen PreparationFFPET specimens are processed and genomic DNA isolated using the cobas® DNA Sample Preparation Kit, a generic manual specimen preparation based on nucleic acid binding to glass fibers. A deparaffinized 5 μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration to be added to the amplification/detection mixture. The target DNA is then amplified and detected on the cobas z 480 Analyzer using the amplification and detection reagents provided in the cobas BRAF Test kit.PCR Amplification and DetectionTarget SelectionThe cobas BRAF Test uses primers that define a 116-base pair sequence of human genomic region containing the BRAF V600E site in exon 15. The entire BRAF gene is not amplified. The test is designed to detect the nucleotide 1799 T>A change in the BRAF gene which results in a valine-to-glutamic acid substitution at codon 600 (V600E). BRAF wild-type and V600E target-specific fluorescent dye-labeled TaqMan probes bind to the wild-type and V600E sequences respectively. The wild-type sequence and the V600E sequence are detected by dedicated optical channels for each sequence.Target AmplificationThermus species Z05 DNA polymerase is utilized for target amplification. First, the PCR reaction mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA Polymerase, in the presence of divalent metal ion and excess dNTPs, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy of the targeted 116-basepair region of the BRAF gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA. Amplification occurs only in the region of the BRAF gene between the appropriate primer pair. The entire gene is not amplified.Automated Real-time DetectionThe cobas BRAF Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, the probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Two different reporter dyes are used to label the target-specific BRAF wild-type (WT; V600) probe and the BRAF V600E mutation probe. Amplification of the two BRAF sequences can be detected independently in a single reaction well by measuring fluorescence at the two characteristic wavelengths.Selective AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas BRAF Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).6 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon due to the use of dUTP as one of the nucleotide triphosphates in the Reaction Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Reaction Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. en The cobas® 4800 BRAF V600 Mutation Test (cobas BRAF Test) is based on two processes: (1) manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET); (2) PCR amplification and detection of target DNA using a complementary primer pair and two oligonucleotide probes labeled with different fluorescent dyes. One probe is designed to detect the wild-type BRAF V600 sequence and one is designed to detect the V600E mutation sequence. Two external run controls are provided and the wild-type allele serves as an internal, full process control.Specimen PreparationFFPET specimens are processed and genomic DNA isolated using the cobas® DNA Sample Preparation Kit, a manual specimen preparation based on nucleic acid binding to glass fibers. A deparaffinized 5 μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration to be added to the amplification and detection mixture. The target DNA is then amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas BRAF Test kit.PCR Amplification and DetectionTarget SelectionThe cobas BRAF Test uses primers that define a 116-base pair sequence of human genomic DNA containing the BRAF codon 600 site in exon 15. The entire BRAF gene is not amplified. The cobas BRAF Test is designed to detect the nucleotide 1799 T > A change in the BRAF gene which results in a valine-to-glutamic acid substitution at codon 600 (V600E). BRAF wild-type and mutant DNA target-specific fluorescent dye-labeled TaqMan probes bind to the wild-type and mutant sequences respectively. The wild-type and mutant sequences are detected by using a dedicated optical channel for each sequence.Target AmplificationThermus species Z05 DNA polymerase is utilized for target amplification. First, the PCR reaction mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA Polymerase, in the presence of divalent metal ion and excess dNTPs, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy of the targeted 116-basepair region of the BRAF gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA. Amplification occurs only in the region of the BRAF gene between the primers.Automated Real-time DetectionThe cobas BRAF Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Two different reporter dyes are used to label the target-specific BRAF wild-type (WT) probe and the BRAF V600E mutation probe. Amplification of the two BRAF sequences can be detected independently in a single reaction well by measuring fluorescence at the two characteristic wavelengths in dedicated optical channels.Selective AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas BRAF Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).13 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon due to the use of dUTP as one of the nucleotide triphosphates in the Reaction Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Reaction Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. en

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