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Nucleic Acids Research, 2001, Vol. 29, No. 16 e77
© 2001 Oxford University Press

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions

Priscilla Wilkins Stevens1,2,*, Jeff G. Hall3, Victor Lyamichev3, Bruce P. Neri3, Manchun Lu4, Liman Wang4, Lloyd M. Smith4 and David M. Kelso1

1Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Science, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107, USA, 2Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4844, USA, 3Third Wave Technologies, 502 South Rosa Road, Madison, WI 53719-1256, USA and 4Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706-1396, USA

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

* To whom correspondence should be addressed at: Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Science, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107, USA. Tel: +1 847 467 2560; Fax: +1 847 491 4928; Email: pwilkins{at}northwestern.edu


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