IDH1/2 RGQ PCR Kit
For the detection of 12 IDH1 and IDH2 mutations in DNA from FFPE brain tumor tissue
The IDH1/2 RGQ PCR Kit enables detection of 12 mutations in the IDH1 and IDH2 genes using real-time PCR on the Rotor-Gene Q 5plex HRM Instrument. The kit provides all necessary reagents, optimized for rapid and sensitive detection of IDH1 and IDH2 mutations.
The IDH1/2 RGQ PCR Kit provides reagents to perform 9 separate amplification reactions for detection of 12 mutations:
Total reaction mixes
The Total Primers and Probe Mixes (PPM-Total) use primers and probes to amplify both mutated and wild-type target sequences.
Mutation detection reaction mixes
The mutation detection primers and probe mixes combine primers and probes, to amplify both mutated and wild-type target sequences, plus an oligonucleotide, 3' blocked with the addition of a phosphate group to prevent elongation (PCR clamping), which is specific to the wild-type target sequence.
When the PCR template contains the wild-type sequence, the 3'-phosphate oligonucleotide will dominate over PCR primer binding due to higher affinity. There is no or low extension by the DNA polymerase and no or low amplification is observed.
When a mutated sequence is present, PCR primer binding will dominate over the 3'-phosphate oligonucleotide binding and amplification will proceed.
Mutation identification reaction mixes
Allele-specific amplification is achieved by ARMS (Amplification Refractory Mutation System), which exploits the ability of the DNA polymerase to distinguish between a match and a mismatch at the 3' end of a PCR primer.
When the PCR primer is fully matched, the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs.
The IDH1/2 RGQ PCR Kit provides all necessary reagents for detection of 12 mutations in the IDH1 and IDH2 genes. Nine primer and probe mixes are used to amplify both mutated and wild-type target sequences. Simply add template DNA and the appropriate primer and probe mix to the ready-to-use PCR master mix, and start the reaction on the Rotor-Gene Q 5plex HRM instrument using the optimized cycling program described in the kit handbook. For DNA purification, we recommend using the QIAsymphony DNA Mini Kit on the QIAsymphony SP or the QIAamp DNA FFPE Tissue Kit, according to the modified protocols in the IDH1/2 RGQ PCR Kit Handbook.
The IDH1/2 RGQ PCR Kit enables detection of 7 mutations in the IDH1 gene, 5 mutations in the IDH2 gene, and identifies 3 major mutations, in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) human brain tumor tissue specimens.
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