|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
42. Single Nucleotide Polymorphism Detection and Identification Directly from Human Genomic DNA by Invasive Cleavage of Oligonucleotide Probes
Victor Lyamichev, Andrea L. Mast, Jeff
G. Hall, James Prudent, Tamara Sander, Monika de Arruda, David Arco, Bruce
P. Neri, and Mary Ann D. Brow
Detection of DNA by invasive cleavage arises from the coordinated action of a pair of overlapping synthetic oligonucleotides hybridized to adjacent regions on a DNA target. At the point of overlap, i.e. where one or more nucleotides from each oligomer compete for same complementary site on the target, a unique secondary structure forms when the 3' end of one oligomer (invasive probe) displaces the 5' end of the other (signal probe). This displaced "flap" is in turn recognized by a structure-specific endonuclease and cleaved to release a fragment.
The specific cleavage of a downstream flap has been employed as an extremely sensitive, quantitative, and highly specific assay for the detection of target DNA both alone and in mixture of extraneous DNA. Because cleavage depends on the correct alignment of the oligonucleotides, the cleavage is sufficiently specific to enable discrimination of single nucleotide polymorphisms (SNPs) and can readily differentiate homo- from heterozygotes in single-copy genes in human genomic DNA. Moreover, we have defined reaction conditions that allow multiple copies of the downstream oligonucleotide probe to be cleaved for each target sequence without temperature cycling, thereby amplifying the cleavage signal and allowing quantitative detection of target DNA at sub-attomole amounts.
The analysis of nucleic acids in this fashion has several advantages over existing methods of oligonucleotide-based detection. First, by requiring two oligonucleotides, the reaction is highly specific for the intended target sequence. Second, the specificity of the enzyme requires precise alignment of the probes for cleavage to occur, providing a much higher level of specificity than can be achieved by hybridization alone, and allowing single-base discrimination of multiple alleles present in a mixed sample. Third, the products of this cleavage reaction can be analyzed via indirect readouts that utilize the nucleotide sequence of products, such as by capture on solid supports, thus simplifying the equipment needed to perform the procedure and adding yet another level of discrimination for the desired cleavage products. Fourth, amplification of a target-dependent signal, rather than the target itself, means that traces of product "carried over" from a completed detection reaction cannot themselves be amplified to lead to false positive results. Finally, detection of specific sequences directly from genomic DNA without intervening DNA amplification avoids false negative or false positive SNP detection that may arise due to low fidelity replication during an amplification step.
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