For in vitro diagnostic use. Others cobas KRAS Mutation Test IVD cobas® KRAS Mutation Test RMD-4800-KRAS-001 05852170190 KIT COBAS 4800 KRAS AMP/DET 24T IVD cobas KRAS Mutation Test 00875197004199 Reagents, kits 1 kit 24 tests false The cobas® KRAS Mutation Test (cobas KRAS Test) is based on two major processes: (1) manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET); and (2) PCR amplification of target DNA using complementary primer pairs and two oligonucleotide probes labeled with fluorescent dye. One probe is designed to detect the KRAS codon 12/13 sequence in exon 2, and the other probe is designed to detect the KRAS codon 61 sequence in exon 3 of the KRAS gene. Mutation detection is achieved by melting curve analysis by the cobas z 480 analyzer. A mutant control, negative control, and calibrator are included in each run to confirm the validity of the run.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 KRAS Test kit.PCR AmplificationTarget SelectionThe cobas KRAS Test kit uses primers that define an 85 base-pair sequence for exon 2 containing KRAS codons 12 and 13 and a 75 base-pair sequence for exon 3 containing KRAS codon 61 in human genomic DNA. Amplification occurs only in the regions of the KRAS gene between the primers; the entire KRAS gene is not amplified.Target AmplificationA derivative of Thermus 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 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 85 base-pair and 75 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. Amplification occurs only in the regions of the KRAS gene between the primer pairs; the entire KRAS gene is not amplified.Automated Real-time Mutation DetectionThe cobas z 480 analyzer is capable of measuring, in real-time, the amount of fluorescence generated by specific PCR products. After amplification, each amplicon generated using the cobas KRAS Test is subjected to a melting program in which the temperature is ramped from 40°C to 95°C (TaqMelt). The wild-type specific probe is bound to both wild-type and mutant amplicon at low temperatures. In the bound state, the fluorescein reporter dye on the 5’ end of the probe is sufficiently far away from the 3’ end quencher dye, allowing the fluorescent dye to emit a specific wave length of light. As the temperature rises, the probe dissociates from the amplicon, allowing the quencher dye to come into close proximity to the fluorescent dye, decreasing the amount of measurable fluorescence. Amplicon with a perfect match to the probe (wild-type) melt at a higher temperature than amplicon with one or more mismatches (mutant). The amount of fluorescence at each temperature increment is measured and the melting temperature(s) are calculated. The presence of a mutant KRAS sequence in exon 2, codons 12 and 13 and in exon 3, codon 61 can be detected when the melting temperatures are within specified ranges. To avoid detection of codon 12 and codon 13 silent mutations (no amino acid change), a modified base serves as a universal base and produces a melting temperature within the wild-type range.Selective AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas KRAS Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).25 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 thymidine triphosphate 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.25 Longo, M.C., Berninger, M.S. and Hartley, J.L. 1990. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 93:125-128. en The cobas® KRAS Mutation Test, for use with the cobas® 4800 System, is a real-time PCR test intended for the identification of mutations in codons 12, 13 and 61 of the KRAS gene in DNA derived from formalin-fixed paraffin-embedded human colorectal (CRC) and non-small cell lung cancer (NSCLC) tissues. en