Abstract
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.
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lyamichev, V., Brow, M. A., and Dahlberg, J. E. (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778–783.
Lyamichev, V., Mast, A. L., Hall, J. G., Prudent, J. R., Kaiser, M. W., Takova, T., et al. (1999) Polymorphism identification and quantita-tive detection of genomic DNA by invasive cleavage of oligonucle-otide probes. Nat. Biotechnol. 17, 292–296.
Kaiser, M. W., Lyamicheva, N., Ma, W., Miller, C., Neri, B., Fors, L., and Lyamichev, V. I. (1999) A comparison of eubacterial and archaeal structure-specific ′’-exonucleases. J. Biol. Chem. 274, 21,387–21,394.
Kwiatkowski, R. W., Lyamichev, V., de Arruda, M., and Neri, B. (1999) Clinical, genetic, and pharmacogenetic applications of the Invader assay. Mol. Diagn. 4, 353–364.
Lyamichev, V. I., Kaiser, M. W., Lyamicheva, N. E., Vologodskii, A. V., Hall, J. G., Ma, W. P., et al. (2000) Experimental and theoreti-cal analysis of the invasive signal amplification reaction. Biochemistry 39, 9523–9532.
Ryan, D., Nuccie, B., and Arvan, D. (1999) Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay. Mol. Diagn. 4, 135–144.
Mein, C. A., Barratt, B. J., Dunn, M. G., Siegmund, T., Smith, A. N., Esposito, L., et al. (2000) Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation. Genome Res. 10, 330–343.
Hessner, M. J., Budish, M. A., and Friedman, K. D. (2000) Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes. Clin. Chem. 46, 1051–1056.
Lyamichev, V., Brow, M. A., Varvel, V. E., and Dahlberg, J. E. (1999) Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain. Proc. Natl. Acad. Sci. USA 96, 6143–6148.
Hall, J. G., Eis, P. S., Law, S. M., Reynaldo, L. P., Prudent, J. R., Marshall, D. J., et al. (2000) Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl. Acad. Sci. USA 97, 8272–8277.
Allawi, H. T. and SantaLucia, J., Jr. (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10,581–10,594.
SantaLucia, J., Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.
About this protocol
Cite this protocol
Lyamichev, V., Neri, B. (2003). Invader Assay for SNP Genotyping. In: Kwok, PY. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 212. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-327-5:229
Download citation
DOI: https://doi.org/10.1385/1-59259-327-5:229
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-968-1
Online ISBN: 978-1-59259-327-9
eBook Packages: Springer Protocols