Description
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: 3 total amplification reactions of codons 132 and 100 of the IDH1 gene and of codon 172 of the IDH2 gene 3 mutation amplification reactions of codons 132 and 100 of the IDH1 gene and of codon 172 of the IDH2 gene 3 mutation-specific amplification reactions of IDH1 R132H, IDH1 R132C, and IDH2 R172K mutations IDH1 and IDH2 mutations detected using the IDH1/2 RGQ PCR Kit Gene Mutation Base change Cosmic ID IDH1 Arg132His 395G>A COSM28746 Arg132Cys 394C>T COSM28747 Arg132Ser 394C>A COSM28748 Arg132Gly 394 C>G COSM28749 Arg132Leu 395G>T COSM28750 Arg132Val 394_395 CG>GT COSM28751 Arg100Gln 299G>A COSM88208 IDH2 Arg172Lys 515G>A COSM33733 Arg172Met 515G>T COSM33732 Arg172Trp 514A>T COSM34039 Arg172Ser 516G>T COSM34090 Arg172Gly 514A>G COSM33731 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.
