Description
The CALR RGQ PCR Kit is intended for real-time PCR on the Rotor-Gene Q 5plex HRM, and includes all required reagents. The kit is optimized for specific and simultaneous identification of the two main CALR mutations (Type 1 & Type 2) and detection of additional minor variants of CALR in genomic DNA, in the same run.
To ensure sensitivity and accuracy, the CALR RGQ PCR Kit has been optimized to perform 7 separate qPCR amplification reactions in a single run for the identification of the two major CALR mutations (Type 1 and Type 2) and the detection of additional minor variants in genomic DNA extracted from human peripheral whole blood.
The CALR RGQ PCR Kit is a ready-to-use kit for the detection of somatic mutations in exon 9 of the gene encoding CALR (cDNA annotation of specific region: c. 1091_1162) (1, 2). The kit uses real-time polymerase chain reaction (PCR) on the Rotor-Gene Q 5plex HRM. To identify Type 1 and Type 2 CALR mutations, an allele-specific amplification is achieved by ARMS (Allele Refractory Mutation System) technology. For the detection of minor variants of CALR mutations, primers and probes to amplify mutated target sequences are combined in the reaction mixes with an oligonucleotide that is 3’-blocked with the addition of a phosphate group which is specific for a wild-type targeted sequence, and prevents elongation of the PCR product (PCR clamping). To validate and control the qPCR reaction in the presence of human genomic DNA template, each CALR reaction mix includes primers and a probe to amplify an endogenous sequence of the ABL1 housekeeping gene. This control sequence is amplified through a multiplex PCR reaction in the presence of all CALR mutant and wild-type DNA. All necessary components are included in the run to provide accurate and sensitive detection of CALR mutations.
The CALR RGQ PCR Kit enables detection of CALR mutations and specific identification of Types 1 and 2 on Rotor-Gene Q 5plex HRM. The first step is DNA extraction, either using the QIAamp DSP DNA Blood Mini Kit (manual) or the QIAsymphony DSP DNA Mini Kit (automated on the QIAsymphony SP), followed by determination of quality and quantity of genomic DNA. The second step is amplification of the genomic DNA by real-time PCR. The kit provides all required reagents to perform 7 separate qPCR amplification reactions in the same run for detection of CALR mutations and identification of Types 1 and 2 in genomic DNA extracted from human peripheral whole blood. Simply start the reaction on the Rotor-Gene Q 5plex HRM using the optimized protocols as described in the kit handbook.
