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TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum , which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC ( n = 46) or NTM ( n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.

The synthesis of a new DNA analogue based on ornithine is described. Only the thymine analogue was synthesised. A 10 mer entirely made up of the thymine monomer forms a triple helix with poly RNA A. The Tm of the triplex was 21°C.

The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide with the linker in conventional phosphoamidite or H-phosphonate DNA syntheses. Directly, or via a post modification step, an oligonucleotide is labelled with one or more reporter moieties, e.g. dansyl (5-dimethylamino)-1-naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and in vitro mutagenesis.

N -(2-benzyloxyethyl)- N -(thymin-1-ylacetyl)glycine was synthesised and used as a linker for coupling DNA and PNA together. Three different 10-mers composed of 6 DNA units and 4 PNA thymine units were made. The PNA-DNA chimera hybridise to complementary DNA or PNA oligomers, and both the PNA and the DNA part of the chimera are involved in the binding.

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-[benzotriazol-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH2, indicated an average yield per synthetic cycle of 97.1%. N1-benzyloxycarbonyl-N6(3)-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the 'low-high' trifluoromethanesulphonic acid procedure.

A synthesis of N -(2-Boc-aminoethyl)- N -(thymin-1-ylacetyl)alanine ( 5 ) is presented. It is demonstrated that chiral integrity can be preserved. PNA (Peptide Nucleic Acid) containing the D-form of 5 hybridizes to complementary DNA with higher affinity than PNA containing the L-form of 5 .

New chromogens expand the colour palette for horseradish peroxidase chromogens used in immunohistochemistry. Tissue staining of cytokeratin with three new cyanine-based chromogens is described. Their use as fluorescent reporters is demonstrated.