Features
- Accurate detection of 7 IDH1 and 5 IDH2 mutations using PCR clamping
- Sensitive identification of 3 mutations using ARMS PCR technology
- A standardized assay with ready-to-use solutions
- An easy workflow with the Rotor-Gene Q instrument
Product Details
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.
Principle
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
| 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.
Procedure
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.
Applications
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.
Resources
Safety Data Sheets (1)
Certificates of Analysis (1)
FAQ
Can I use stained slide materials with IDH 1/2 RGQ PCR kit?
Can I perform the IDH 1/2 RGQ PCR test with slides containing less than 40% tumor cells?
Can I use the QIAGEN deparaffinization solution with the IDH 1/2 RGQ PCR kit?