Binary deoxyribozyme probe for highly sensitive nucleic acid analysis
Lead Inventors: Dmitry Kolpashchikov, Ph.D.Problem or Unmet Need:To date, there is only a limited repertoire of diagnostic assays available that allow for the surveillance of a few or single base pair variability in a nucleic acid target sequence. Assays based on PCR have the potential to meet this need but suffer from time consuming protocols that cannot be implemented at physiological conditions relevant for in vivo applications. Non PCR based technologies lack sensitivity, yielding them unusable for very low levels of analyte. Diagnostic assays for the presence of a specific nucleic acid sequence require a fast, sensitive and highly selective technology that can be applied at physiological conditions. This technology is a non-PCR based nucleic acid detection system. The approach is based on the use of a deoxyribozyme that consists of two chemically stable and inexpensive DNA strands that are dissociated in the absence of the nucleic acid analyte, yielding the deoxyribozyme inactive. The strands hybridize in adjacent positions when they encounter their nucleic acid target and restore the DNA enzyme catalytic core. The active deoxyribozyme cleaves a fluorophore and quencher labeled oligonucleotide substrate or triggers cascades of deoxyribozymes that amplify the fluorescence signal created by the labeled substrate cleavage.
The method is highly selective allowing for reliable discrimination of single base substitutions at any position in the analyte sequence Due to the unique signal amplification technique, the detection system is extraordinarily sensitive and has the potential to detect low concentrations of nucleic acids in sample The technology can be implemented at room temperature and in mild physiological conditions allowing for inexpensive and flexible application The variable components of this system are easy to make and inexpensive, the additional reagents are uniform for any analyte sequence and allow for low cost multiplex application
The technology can be used in diagnostic assays detecting specific viral and bacterial RNAs The method can be applied in genotyping single nucleotide polymorphisms The technology has the potential to be used in clinical and laboratory assays both in vitro and in the cell for single or few base pair mutations in a sequence of interest
This technology is a non-PCR based nucleic acid detection system. The approach is based on the use of a deoxyribozyme that consists of two chemically stable and inexpensive DNA strands that are dissociated in the absence of the nucleic acid ana...
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