For life science research only. Not for use in diagnostic procedures. Others BRAF NRAS Mutation Test LSR LSR BRAF/NRAS Mutation Test (LSR) RMD-4800-LSR-001 07659962001 BRAF/NRAS Mutation Test (LSR) BRAF/NRAS Mutation Test (LSR) 07613336100523 Reagents, kits 24 reactions Not Available false Target selectionThe BRAF/NRAS Mutation Test (LSR) uses primers that define specific base-pair sequences for each of the targeted mutations. Amplification occurs only in the regions of the BRAF or NRAS genes between the primers; the entire gene is not amplified. BRAF sequences range from 101 – 120 base pairs. NRAS sequences range from 94 – 121 base pairs.Target amplificationA derivative of Thermus species Z05-AS1 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 dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double stranded DNA copy which includes the targeted base-pair regions of the BRAF and NRAS genes. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.Automated real-time mutation detectionThe BRAF/NRAS Mutation Test (LSR) kit 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-AS1 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. Three different reporter dyes are used to label the mutations targeted by the test. Amplification of the targeted BRAF and NRAS sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.Selective amplificationSelective amplification of target nucleic acid from the sample is achieved in the BRAF/NRAS Mutation Test (LSR) kit by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). 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 in place of deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; 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 Master Mix reagents, 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 BRAF/NRAS Mutation Test (LSR) is an allele-specific, real-time PCR test for the qualitative detection and identification of exon 11 and 15 mutations in the proto-oncogene B-Raf (BRAF) gene and exon 2, 3, and 4 mutations in the neuroblastoma RAS viral oncogene homolog (NRAS) gene from formalin-fixed, paraffin-embedded tissue (FFPET) or plasma samples. It is intended for life science research only and is not for use in diagnostic procedures. en