Analytical method and kit
AssigneeThe Secretary of State for Defence
InventorsDavid James Squirrell, Martin Alan Lee
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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.
Claims12
A method for detecting the presence or amount of a target DNA sequence within a sample, said method comprising conducting a DNA amplification reaction, which uses at least one primer, in the presence of (a) an RNA probe which is specific for at least a portion of said target DNA sequence downstream of the primer;
- (b) an enzyme which hydrolyses RNA when in the form of an RNA/DNA duplex and (c) one or more enzymes and/or reagents necessary to convert adenosine monophosphate produced to adenosine triphosphate (ATP);
- adding to the sample bioluminescent reagents which react to the presence of ATP, detecting a signal from said bioluminescent reagents and relating that to the presence or amount of the target DNA sequence.
A method of detecting a target DNA sequence in a sample, wherein the method comprises
- amplifying a target DNA sequence with a forward and reverse primer via polymerase chain reaction, wherein during amplification an RNA probe that comprises a complementary sequence to a region in the target DNA sequence being amplified is hybridized, and the hybridized RNA probe is hydrolyzed, producing adenosine monophosphate,
- converting the adenosine monophosphate to adenosine triphosphate with an enzyme, and
- detecting the presence of the target DNA sequence by a bioluminescence reaction.
Dependencies are inferred from the claim text.