Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-05-20
1998-10-27
Sisson, Bradley L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 435810, 436164, 436805, 252700, 536 243, 536 266, 935 8, 935 77, 935 78, C12Q 168, C09K 300, C07H 2104, C12N 1500
Patent
active
058276530
DESCRIPTION:
BRIEF SUMMARY
A method is disclosed for the detection of analytes such as DNA or RNA sequences. The method involves forming a complex comprising the analyte and a complementary binding entity such that, for DNA or RNA, hybridisation occurs to form a duplex. Observation of the matching complex is obtained by adding a third, sensitising component that either intercalates or groove binds with the duplex sequence (the duplex binder). The complementary binding entity is designed to incorporate a lanthanide ion and the duplex binder incorporates a ligand for the lanthanide ion that can also act as a sensitiser. Alternatively the complementary binding entity can be attached to the sensitiser and the duplex binder to the lanthanide ligand. When binding in the collaborative manner is achieved, the three component system is irradiated with light which can be selectively absorbed by the sensitiser. The excited sensitiser can donate its energy to the lanthanide ion by a direct ligand to metal energy transfer mechanism such that the lanthanide ion becomes excited. Subsequent emission of light from the excited lanthanide ion signals the formation of the complex and hence the presence of the analyte. Luminescence from the lanthanide species is characterised by a long lifetime, involving a delayed emission process, which can be measured after decay of background fluorescence signals. The method may be applied to either heterogeneous or, preferably, homogeneous assays and may be used either qualitatively or quantitatively.
Methods for the in vitro detection of analytes are well known in the art. The methods include the formation of antibody-antigen complexes, as in immunoassays, and the formation of nucleic acid complexes, as in polynucleotide hybridisation. Polynucleotide hybridisation assays using a polynucleotide probe for verifying the presence of a target polynucleotide target is a well known method. Polynucleotide hybridisation is based upon the ability of a DNA or RNA sequence to form complexes with a complementary dna or rna strand. When single stranded polynucleotide probes are incubated in solution with single stranded target molecules under defined conditions, complementary base sequences-pair to form double-stranded hybrid molecules. Such hybridisation can occur in solution. Alternatively, either the target strand or the probe may be immobilised on a support in which case hybridisation leads to double stranded hybrid molecules which are thus immobilised. In this case any unbound polynucleotide molecules may be washed off whilst leaving the Grunstein and J. Wallis, Methods in Enzymology, 1979, 68, pp. 379-469; A. R. Sambrook, Methods in Enzymology, 1980, 65 (part 1), pp. 468-478; `Modified Nucleotides and Methods of Preparing and Using the Same`, by D. C. Ward, A. A. Waldrop and P. R. Langer, European Patent Publication, 063,879; `DNA Probes for Infectious Disease` by A. J. Berry, and J. B. Peter, Diagnostic Medicine, 1984, March, pp. 1-8.!
The polynucleotide probes comprise a polynucleotide segment and a latent signalling segment which is attached to the polynucleotide. The polynucleotide segment of the probe has the ability to hybridise (base pair) to a complementary sequence of interest within the target polynucleotide (the target). The latent signalling portion of the probe produces the means by which the presence of the analyte can be verified. Whilst methods can involve, for example, fluorescence, phosphorescence, radioactivity, chromogen formation or electron density, this application concerns the use of delayed luminescence.
The method for detecting the presence of a target polynucleotide generally involves several steps, one of which involves the separation of hybridised polynucleotide probe from the unhybridised probe or mismatched target, as in heterogeneous or sandwich type assays. Typically, double stranded polynucleotides are isolated from a sample suspected of containing a target polynucleotide sequence. The double stranded polynucleotides are cut into smaller segments by means of restriction endonuclease enzym
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Garman Andrew John
Sammes Peter George
Sisson Bradley L.
Zeneca Limited
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