Project Leader: Dr Dave Bunyan

Company website: www.twt.com (Third Wave™ Technologies)

Invader® assay technology:

The Invader® DNA assay enables simultaneous detection of two different alleles in a bi-allelic system in a non-PCR based assay. Two oligonucleotide probes (an allele specific primary probe and an Invader® probe) hybridise in tandem to the target DNA to form a specific overlapping structure. The 5’-end of the primary probe contains a 5’-Flap that is non-complementary to the target DNA and so is unable to hybridise to the target sequence. The 3’-end of the bound Invader® probe overlaps the primary probe by a single base this is the site of the allelic variant or SNP.
A Cleavase® enzyme (Flap endonuclease I) recognises the overlapping structure and cleaves the 5’-Flap on the primary probe at the base of the overlap releasing it as a target specific product. If the probe does not hybridise perfectly at the site of interest no overlapping structure is formed and no cleavage occurs and the target specific product is not released. The reaction consists of two primary probes and two invader probes that are specific to either allele 1 or allele 2 of a bi-allelic system meaning two target specific products are generated.

The target specific 5’-Flap oligos’s are involved in a secondary reaction where they act as Invader® probes on a Fluorescent resonance energy transfer (FRET™) cassette leading to the formation of an overlapping structure that is recognised by the Cleavase® enzyme. When the FRET™ cassette is cleaved a fluorophore is released form a quencher on the FRET™ generating a fluorescence signal. There are two signal fluorophores attached to two different FRET™ cassettes (FRET™ 1 and FRET™ 2) that are spectrally distinct and specific to either allele of the bi-allelic system. The ratios of the two fluorescent signals then allows a genotype to be assigned.

The reaction conditions (63^oC) allow cycling of the primary probes and Invader® probes meaning multiple rounds of primary probe cleavage occurs per DNA target and multiple fluorescent signals are generated per DNA target.

Detection of Factor V Leiden (G1691A), Factor II (G20210A ) & MTHFR (C677T) variants:

The Invader® assay was evaluated in conjunction with other SNP detection technologies (Pyrosequencing™ and NanoChip®) by testing 100 anonymised thrombophilia patients samples for the Factor V Leiden (G1691A), Factor II (G20210A ) & MTHFR (C677T) variants that had previously been genotyped using restriction fragment length polymorphism (PCR based assay). The FVL and FII results display 100% concordance to the RFLP method and testing of the MTHFR variant is currently being undertaken.

Evaluation of the Invader® assay platform for molecular analysis of the Factor V (G1691A) and Factor II (G20210A) mutations. Presentation to the CMGS Spring Meeting in 2004 available for download here.

Other applications of the Invader® assay platform: