For life science research only. Not for use in diagnostic procedures. Others KRAS Mutation Test v2 LSR LSR KRAS Mutation Test v2 (LSR) RMD-4800-LSR-002 KRAS Mutation Test v2 (LSR) 07613336103821 1 kit false KRAS Mutation Test v2 (LSR) 07989270001 24 reactions Reagents, kits en The KRAS Mutation Test v2 (LSR) is an allele-specific, real-time PCR test for the qualitative detection and identification of exon 2, 3, and 4 mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene from formalin-fixed, paraffin-embedded tissue (FFPET) and from cfDNA derived from plasma.It is intended for life science research only and is not for use in diagnostic procedures. en Target selectionThe KRAS Mutation Test v2 (LSR) uses primers that define specific base-pair sequences for each of the targeted mutations. Amplification occurs only in the regions of the KRAS gene between the primers; the entire gene is not amplified. An endogenous Internal Control (IC) is amplified in each sample to verify reagent and DNA quality. The IC detects a region at the 3’ end of the KRAS gene that does not typically experience somatic mutations. The targeted KRAS sequences range from 79 – 114 base pairs.Target amplificationA derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR 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 cation 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 KRAS gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.Automated real-time mutation detectionThe KRAS Mutation Test v2 (LSR) 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. Four different reporter dyes are used to label the mutations targeted by the test. Amplification of the targeted sequences is 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 KRAS Mutation Test v2 (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.