Qiagen, 873001, IDH1/2 RGQ PCR Kit (20)

For 20 reactions: 9 Primer and Probe Mixes, WT Control, Positive Control, Master Mix, Nuclease-Free Water

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

Principle

Gene: Mutation
IDH1: Arg132His
Arg132Cys: 394C>T
Arg132Ser: 394C>A
Arg132Gly: 394 C>G
Arg132Leu: 395G>T
Arg132Val: 394_395 CG>GT
Arg100Gln: 299G>A
IDH2: Arg172Lys
Arg172Met: 515G>T
Arg172Trp: 514A>T
Arg172Ser: 516G>T
Arg172Gly: 514A>G

- 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

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.