Nucleic acid detection method

Abstract

The present invention provides a method of detecting a target nucleotide sequence in a nucleic acid molecule, which comprises: (a) binding of an oligonucleotide probe to said nucleic acid molecule; (b) selective labelling of the bound oligonucleotide probe in the presence of said target nucleotide sequence; (c) hybridization of the labelled oligonucleotide to a complementary sequence; and (d) subsequent detection of the label. Such methods are suitable for qualitative and quantitative assays of microbiological populations.

21 . A kit for carrying out a method of detection of a target nucleotide sequence in a nucleic acid molecule which comprises: 
 (a) an oligonucleotide probe capable of binding to a target nucleic acid molecule containing the target nucleotide sequence;    (b) means for selective labelling of the oligonucleotide probe; and    (c) a nucleotide sequence complementary to the oligonucleotide probe.    
     
     
         22  A kit as claimed in  claim 21  wherein the nucleotide sequence of (c) is fully complementary to the oligonucleotide probe.  
     
     
         23  A kit as claimed in  claim 21  wherein the oligonucleotide probe is 20 to 30 nucleotides in length.  
     
     
         24  A kit as claimed in  claim 21  wherein the means (b) for selective labelling of the oligonucleotide probe provides for incorporation of a labelled nucleotide.  
     
     
         25  A kit as claimed in  claim 24  wherein means (b) for selective labelling of the oligonucleotide probe comprises a labelled nucleotide.  
     
     
         26  A kit as claimed in  claim 25  wherein the labelled nucleotide is a labelled dideoxynucleotide.  
     
     
         27  A kit as claimed in  claim 24  wherein the means for selective labelling of the oligonucleotide probe comprises one or more labelled dideoxynucleotides and one or more unlabelled dideoxynucleotides.  
     
     
         28  A kit as claimed in  claim 27  wherein the means for selective labelling of the oligonucleotide probes comprises one labelled dideoxynucleotide and three unlabelled dideoxynucleotides.  
     
     
         29  A kit as claimed in  claim 21  wherein the oligonucleotide probe is designed with one or more mismatches at the 3′-end to non-target nucleotide sequences.  
     
     
         30  A kit as claimed in  claim 21  wherein the sequence complementary to the labelled oligonucleotide is immobilised on a solid support.  
     
     
         31  A kit as claimed in  claim 30  wherein the solid support is a membrane strip or nucleic acid chip.  
     
     
         32  A kit as claimed in  claim 21  further comprising (d) means to amplify the nucleic acid molecule which contains the target sequence.  
     
     
         33  A kit as claimed in  claim 32  which further comprises a competitor nucleic acid molecule for coamplification with the nucleic acid molecule which contains the target sequence.  
     
     
         34  A kit as claimed in  claim 33  wherein the competitor molecule comprises a recognition sequence which is complementary to a competitor oligonucleotide probe.  
     
     
         35  A kit as claimed in  claim 33  further comprising a competitor oligonucleotide probe.  
     
     
         36  A kit as claimed in  claim 35  wherein the competitor oligonucleotide probe is capable of being selectively labelled after hybridisation to the competitor molecule.  
     
     
         37  A kit as claimed in  claim 36  further comprising the means to detect said labelled competitor oligonucleotide probe.  
     
     
         38  A kit as claimed in  claim 37  wherein the sequences which are complementary to the oligonucleotide probe are immobilised on a solid support in discrete, pre-determined positions.  
     
     
         39  A kit as claimed in  claim 21  wherein the target nucleotide sequence is characteristic of a particular organism or group of organisms.  
     
     
         40  A kit as claimed in  claim 39  which comprises a plurality of different oligonucleotide probes, each probe species being capable of binding to a different target nucleotide sequence, each target sequence being characteristic of a particular organism or group of organisms.