Analytical Method and Kit

Abstract

Analytical methods using RNA probes for the detection or analysis of nucleic acid sequences is described. These probes are contacted with a sample suspected of containing the nucleic acid sequence and if they form duplexes, they are hydrolysed. This may be done, for example during an amplification reaction. AMP generated as a result of the hydrolysis is converted to ATP. The ATP may then be detected using bioluminescent reagents.

1 - 35 . (canceled) 
   
   
       36 . A method for detecting the presence or amount of a target nucleic acid within a sample, said method comprising denaturing nucleic acids within the sample; contacting these nucleic acids with an RNA hydrolysis probe which is specific for at least a portion of said target nucleic acid so that the probe forms duplexes with the target nucleic acid; adding an RNAse enzyme which hydrolyses RNA only when in double stranded form and one or more enzymes or reagents necessary to convert adenosine monophosphate produced to adenosine triphosphate; adding to the sample bioluminescent reagents which react to the presence of ATP; detecting a signal from said bioluminescent reagents; and, relating that signal to the presence or amount of the target nucleic acid sequence. 
   
   
       37 . The method of  claim 36 , wherein the one or more enzymes or reagents necessary to convert adenosine monophosphate to adenosine triphosphate comprises
 a) phosphoenolpyruvate synthase, phosphate and phosphoenlpyruvate; and/or   b) nucleoside triphosphate-adenylate kinase, nucleoside 5′-triphosphate (NTP) and adenylate kinase; and/or   c) pyruvate phosphate dikinase.   
   
   
       38 . The method of  claim 36 , wherein the bioluminescent reagents comprise luciferin and luciferase. 
   
   
       39 . The method of  claim 36 , wherein the bioluminescent reagents further comprise a source of magnesium ions. 
   
   
       40 . The method of  claim 36 , further comprising conducting an amplification reaction, wherein the target nucleic acid is DNA and the reaction is conducted in the presence of a DNA polymerase. 
   
   
       41 . The method of  claim 40 , wherein the bioluminescent reagents are present or added throughout the amplification reaction. 
   
   
       42 . A method for determining the sequence of a nucleic acid, said method comprising
 a) binding an RNA probe to a known region of said sequence such that at least one nucleotide at an end of said probe reaches into an unknown or uncertain region of the sequence;   b) hydrolysing the RNA probe using an RNAse enzyme which hydrolyses RNA only when in double stranded form;   c) converting adenosine monophosphate produced to adenosine triphosphate;   d) adding to the sample bioluminescent reagents which react to the presence of ATP;   e) detecting a signal from said bioluminescent reagents; and   f) relating that signal to the presence of a region of the sequence which is complementary or otherwise to the end of the probe.