There is increasing recognition that mutations associated with cancer can be used as prognostic or predictive biomarkers. Polynucleotide Assisted Sequence Switching (PASS) primers are a novel technology capable of sensitively & specifically detecting highly similar targets like SNPs and somatic mutations such as gene insertions, deletions and point mutations, with sensitivity up to 0.1% in a multiplex reaction.
PASS primers consist of target specific regions separated by an insert sequence (IS) which is not complementary to the target region. A long 5’ target specific region anchors the primer and a short 3’ region targeting the variant base/s directs specific binding and extension. After amplification, the IS sequence is inserted into the amplicon and acts as a ‘barcode’ to identify the amplicon or genetic variation when coupled with MNAzyme® qPCR.
MNAzymes (Multi-component Nucleic Acid enzymes) are a unique real-time detection technology made up of nucleic acid components. The active MNAzyme forms when bound to target, and cleaves a fluorescently labelled probe generating a real-time signal. Superior multiplexing capacity is enabled by coupling PASS with MNAzyme® qPCR. Each multiplexed target is given a distinct IS and this ‘barcode’ is specifically detected by the MNAzyme.
This technology has been applied to the multiplex detection of ADME SNPs for drug absorption, distribution, metabolism, and excretion, demonstrating equivalent results to TaqMan duplexes, while providing an additional advantage in enabling multiplex tri-allelic SNP detection.
PASS MNAzyme® qPCR has been used to develop multiplex systems for the detection of 13 KRAS codon 12, 13 & 61 mutations with sensitivities of <1% of mutant allele. The performance of the assay was evaluated against characterised colon cancer and melanoma FFPE samples demonstrating >96% concordance with Sequenom MassARRAY and Illumina MiSeq.
PASS MNAzyme® qPCR technology affords both the advantage of sensitivity and specificity for the multiplex detection of cancer targets.