The type of TaqMan technology used in the kits is based on destructible oligonucleotide probes with chemical modifications of locked nucleic acids and has a high sensitivity and specificity. Depending on the total amount of DNA in the sample, there is up to 1% of mutant against a background of wild DNA determined with an accuracy of at least 99,9%. The features of the kits enable reproductive detection of mutations occurred in the sample even in low concentration minimizing probability of false-negative results.
We have developed the kits for such oncogenes as EGFR, KRAS, BRAF,etc. and conducted their trials in Russian laboratories. The kits are compatible with all common real-time PCR thermocyclers: ABI7300/7500/StepOne Bio-Rad iQ5/CFX, Rotor-Gene 3000/6000, DТ-prime and etc.
Two DNA probes are added to the reaction mixture (complementary to wild and mutant DNA), each of which contains a fluorescent label (usually FAM and VIC/HEX) and a fluorescence quencher. The initial solution has a minimal fluorescence background, but in the result of amplification the fluorescent dyes are released and this equates to detect the presence of mutant DNA sample in the amplified mixture. Special amplification conditions and probe design allow to achieve high specific detection of mutant DNA in a single reaction tube.
In comparison with analogues the applied technology has a wide range of DNA sufficient concentration (from 0.5 ng to 300 ng per reaction) and allows considerable reduction of test time, because there is no need for quantitative calibration of samples, and also eliminates false positive results, which are typical for allele-specific PCR in case of DNA excess.