therascreen KRAS RGQ PCR Kit

The therascreen KRAS RGQ PCR Kit is intended to detect 7 mutations in codons 12 and 13 of the KRAS gene. The kit utilizes two technologies — ARMS and Scorpions — for detection of mutations in real-time PCR.

ARMS

Allele- or mutation-specific amplification is achieved by ARMS (Amplification Refractory Mutation System). ARMS primers preferentially anneal with DNA containing the mutation and allow Taq DNA polymerase to initiate PCR, effectively distinguishing between a match and a mismatch at the 3" end of a PCR primer. Specific mutated sequences are selectively amplified, even in samples where the majority of the sequences do not carry the mutation. When the primer is fully matched, the amplification proceeds with full efficiency. When the 3" base is mismatched, only low-level background amplification occurs. 

Scorpions

Detection of amplification is performed using Scorpions. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a probe. The technology uses a fluorescence-based method to indicate the presence of the mutation. The Scorpion primer hybridizes with a DNA sequence upstream of the target region. The primer is then extended by Taq DNA polymerase and the target region is copied. The newly copied region is complementary to the probe region of the Scorpion. Following a temperature increase within the real-time PCR cycler, the extended Scorpions primer denatures. When the solution cools, the Scorpions probe self hybridizes. The fluorophore is separated from the quencher and a fluorescence signal is generated.

Extract DNA samples from formalin-fixed paraffin-embedded (FFPE) colorectal cancer tumor tissue collected from colorectal cancer patients using the QIAamp DSP DNA FFPE Tissue Kit, which has been validated for use with the therascreen KRAS RGQ PCR Kit. The therascreen KRAS RGQ PCR Kit uses a two-step procedure. The first step is performance of the control assay to assess the total amplifiable DNA in a sample. The second step is to test with the mutation assays in order to detect the presence or absence of KRAS mutations.

The therascreen KRAS RGQ PCR Kit enables the detection of 7 mutations in codons 12 and 13 of the human KRAS gene using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue samples. The therascreen KRAS RGQ PCR Kit is only intended to discriminate between KRAS mutation-negative (wild-type) and KRAS mutant tumors. KRAS mutations detected by the therascreen KRAS RGQ PCR Kit include:

• 12ALA
• 12ASP
• 12ARG
• 12CYS
• 12SER
• 12VAL
• 13ASP