Non-gel based SNP-detection using Varioskan and the Invader® Technology

Abstract

Single-nucleotide polymorphism (SNPs) are the most frequently found polymorphisms in genomes and have been promoted as the genetic markers of choice for the research of complex genetic features (1).

There are several systems for detecting SNP’s. Most are PCR-based and usually need gel-electrophoreses. The Invader technology is a good alternative as it is a non-PCR and non-gel based detection system.

The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. This means that Invader is based on signal rather than product amplification (2, 3). FRET reporter system in the Invader technology uses individual FRET pair for each allele. Each FRET pair is formed by a fluorescent label and label specific fluorescence quencher. Each Invader probe is allele-specific and the combination of results gives the genotype of the sample. For more information about Invader technology, see http://www.twt.com/.

In this application note the Invader assay products (unknown and control samples) were measured by Thermo Varioskan. By using monochromators in stead of filters, Varioskan makes it possible to use a variety of different FRET labels and even multiplex them. In this experiment, a biplex format with two FRET pairs was used, FAM and RED both connected to their specific quenchers.