Amplifying nucleic acid sequences in samples containing other sequences to be suppressed, e.g. in diagnostic tests, with addition of new or known discriminating oligomers of locked nucleic acid type

摘要

Amplifying nucleic acid sequences (I) to be detected in a sample also containing other nucleic acid sequences (II) to be suppressed (where (I) and (II) differ in at least one base but correspond in many bases), comprising adding a discriminating oligomer (III) (which binds to (II) but not to (I)) to the amplification reaction mixture, is new. Amplifying nucleic acid sequences (I) to be detected in a sample also containing other nucleic acid sequences (II) to be suppressed (where (I) and (II) differ in at least one base but correspond in many bases) is carried out using a discriminating oligomer (III) (formed from an appropriate sequence of monomers) which (at a suitable, selected amplification reaction temperature) can undergo base pairing with (II) and binds to (II) but not to (I), is new. The process involves: (a) adding a suitable concentration of (III) to the enzymatic or chemical amplification reaction mixture, so that (III) binds to (II) but not to (I); (b) carrying out the amplification reaction enzymatically or chemically using appropriate reagents; and (c) determining the presence of amplification products of (I). The novel feature is that (III) consists at least partially of ribonucleoside residues in which the O-atom at the C2 carbon of the ribose is covalently bonded to the C4 carbon via methylene, oxygen, sulfur or secondary amino. Independent claims are included for: (1) a corresponding method for detecting (I) in a sample also containing (II), based on bonding a fluorescent dye-labeled oligonucleotide with (I) and determining the consequent change in fluorescence intensity, where a discriminating oligomer (III) as described above is added to the sample; (2) new oligomers (III') formed from monomers at least partially containing ribonucleoside residues in which the O-atom at the C2 carbon of the ribose is covalently bonded to the C4 carbon via methylene, oxygen, sulfur or secondary amino, where (III') additionally contain covalently bonded residue(s) of a molecule (IV) which (due to its chemical nature) can interact with DNA, RNA or double stranded hybrids of DNA and RNA via ionic or nonionic interactions not corresponding to the base pairing of natural DNA or RNA; and (3) kits for carrying out nucleic acid sequence amplification reactions, which include (III) as defined above.