Invader Assay for SNP Genotyping
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In the basic Invader assay, two synthetic oligonucleotides, the invasive and signal probes, anneal in tandem to the target strand to form the overlapping complex shown schematically as primary reaction in Fig. 1 . The signal probe is designed to have two regions: a target-specific region that is complementary to the target sequence, and a 5′ arm or flap that is noncomplementary to both the target and the invasive probe sequences. Structure-specific 5′ nucleases (1 ), known as Cleavase enzymes, recognize this overlapping complex and cleave the signal probe at the site of its overlap with the 3′ end of the invasive probe, as shown by the arrow in Fig. 1 (2 –4 ). This cleavage releases the noncomplementary 5′ flap of the signal probe plus one nucleotide of its target-specific region. The cleaved 5′ flap serves as a signal for the presence, and enables quantitative analysis, of the specific target in the sample.
Fig. 1. Invader assay for analysis of the (A) C677 and (B) T677 polymorphisms in the human methylenetetrahydrofolate reductase (MTHFR) gene. The sense strand of the MTHFR gene was used for the analysis. The invasive and primary (signal) probes overlap by one nucleotide at the polymorphic site (shown in bold case). The primary probes have two regions: the target-specific region and the non-complementary 5′ flap. The overlapping complex is specifically cleaved by a structure-specific 5′ nuclease at the site of the overlap (shown by arrows) releasing the 5′ flap of the primary probe. In the secondary reaction, the cleaved 5′ flap from the primary reaction forms an overlapping complex with the FRET cassette. The FRET cassette for the C677 allele includes the FAM (F) dye and Z21 quencher, while the FRET cassette for the T677 allele includes Redmond Red− (R) and the Z21 quencher. The cleavage sites of the FRET cassettes in the presence of cleaved 5′ flaps are shown by arrows.
