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IDH1/2 RGQ PCR Kit

For the detection of 12 IDH1 and IDH2 mutations in DNA from FFPE brain tumor tissue
  • 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
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.
Cat No./ID: 873001
IDH1/2 RGQ PCR Kit (20)
For 20 reactions: 9 Primer and Probe Mixes, WT Control, Positive Control, Master Mix, Nuclease-Free Water
For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
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

 

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.

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.
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