SEQUENCE-SPECIFIC DETECTION OF NUCLEIC ACID HYBRIDS USING A DNA-BINDING MOLECULE OR ASSEMBLY CAPABLE OF DISCRIMINATING PERFECT HYBRIDS FROM NON- PERFECT HYBRIDS

摘要

Method for detecting or localizing specific nucleic acid sequences with a high degree of sensitivity and specificity which comprises: (a) adding PNAs containing a 1/2 BBR and a 1/2 TBR to a sample containing or suspected of containing TNAs containing 1/2 TBR sequences, to form a complex having target binding regions, TBRs, formed by the hybridization of complementary 1/2 TBRs present in the PNAs and TNAs respectively; (b) binding the TBRs formed in step (a) to a TBA to form a TBA-TNA-PNA complex, provided that said binding may occur prior to, during or subsequent to said hybridization of step (a); (c) adding Booster Nucleic Acids, BNAs, containing booster binding regions, 1/2 BBRs, to the complex formed in step (b) such that the 1/2 BBRs in the BNAs hybridize with the 1/2 BBR sequences present in the PNAs or to 1/2 BBRs present in BNAs already bound to the PNA, to form BBRs, such that TBA-TNA-PNA- (BNA)n complexes are formed: (d) adding Hairpin Nucleic Acids, HNAs, 2584 י" ב בתשרי התשס" ה - September 27, 2004 containing 1/2 BBR sequences, to the complex formed in step (c) such that the 1/2 BBRs in the HNAs hybridize with any available 1/2 BBR sequences present in the BNAs of the complex of step (c), thereby capping the extension of the BNAs onto the TBA-TNA-PNA-BNA-(BNA)n complexes: (e) adding Booster Binding Assemblies, BBAs, linked to indicator moieties, to the TBA-TNA-PNA-(BNA)n-HNA complexes formed in step (d) to form TBA-TNA- PNA-(BNA-BBA)n-HNA complexes, provided that said BBAs are added prior to, concurrent with or subsequent to addition of said BNAs of step (c); and (f) detecting the signals produced by the indicator moieties linked to the TBAs, PNAs, BBAs or HNAs in the TBA- TNA-PNA-(BNA-BBA)n-HNA complexes of step (e): wherein the TNA comprises: (i) one or more specific 1/2 TBR sequences, the presence or absence of which in a particular sample is to be confirmed: the PNA comprises: (i) a single-stranded sequence, 1/2 TBR, which is capable of forming, under hybridizing conditions, a hybrid, TBR, with a 1/2 TBR present in target nucleic acid (TNA); (ii) a single stranded sequence, 1/2 BBR, which is capable of forming, under hybridizing conditions, a hybrid BBR with a 1/2 BBR present in a booster nucleic acid (BNA); and (iii) an OSA, which has no attached support and/or indicator, or an attached support or indicator or both selected from the group consisting of attachment to beads, polymers, proteins, peptides and surfaces, and/or indicators: the BNA comprises: (i) a 1/2 BBR, as shown in Fig. 1(IIb), as given in the specification, which has a sequence which is complementary to a 1/2 BBR sequence in a PNA and which is capable of forming, under hybridizing conditions, a hybrid, BBR, with the PNA; (ii) an OSA, which has no attached support or indicator, or is an attached support or indicator or both selected from the group consisting of attachment to beads, polymers, proteins, peptides and surfaces, and/or indicators; (iii) additional hybridization sites, 1/2 BBRs, for other BNAs; and (iv) sequences, 1/2 BBRs, which can hybridize to BNAs already hybridized to the PNA: the BBA comprising: (i) a molecule or assembly, or a portion of a molecule or assembly which is capable of selectively binding to a BBR; and (ii) has no attached support and/or indicator, or an attached support or indicator or both which is selected from the group consisting of attachment to beads, polymers, proteins, peptides and surfaces, and/or indicators.