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US 2002/0119450 A1Published application
Fluorimetric detection system of a nucleic acid
AssigneeThe Secretary of State for Defence
InventorsMARTIN A LEE, RODERICK FUERST
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AssigneeThe Secretary of State for Defence
InventorsMARTIN A LEE, RODERICK FUERST
Estimating the term…
A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising: (a) adding to a sample suspected of containing said target nucleic acid sequence, a probe specific for said target sequence and DNA duplex binding agent, said probe comprising a reactive molecule able to absorb fluorescence from or donate fluorescent energy to said DNA duplex binding agent, (b) subjecting the thus formed mixture to an amplification reaction in which target nucleic acid is amplified, (c) subjecting said sample to conditions under which the said probe hybridises to the target sequence, and (d) monitoring fluorescence from said sample. This method can be used for example to monitor amplification reactions such as PCR reactions, such that the amount of target sequence present in the sample may be determined. Additionally or alternatively, it may be used to generate duplex destabilisation data such as melt hysteris information for amplification monitoring or for detection and quantitation of polymorphisms or allelic variation, and so is useful in genetic diagnosis.
A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising,
A method according to any one of the preceding claims wherein the DNA duplex binding agent is an intercalating dye.
A method according to any one of the preceding claims wherein the target nucleic acid is rendered single stranded prior to hybridization to the probe in step (c).
A method according to any one of the preceding clam wherein the amplification reaction is the polymerase chair reaction (PCR).
A method according to any one of the preceding claims wherein the probe hybridises with the target nucleic acid during even cycle of the amplification reaction.
A method according to any one of the preceding claims wherein the fluorescence from both the dye and the reactive molecule are monitored.
A method according to any one of the preceding claims wherein the reactive molecule is a rhodamine dye, Cy5 or fluorescein.
A method according to any one of the preceding claims wherein the reactive molecule is attached at an end region of the probe.
A method according to any one of he preceding claims wherein the probe is designed such that it is hydrolysed by the DNA polymerase used in the amplification reaction.
A method according to any one of the preceding claims wherein the amplification reaction is effected using 5′-3′ exonuclease lacking enzyme.
A method according to any one of the preceding claims wherein the 3′ end of the probe is blocked to inhibit extension thereof during the extension phase.
A method according to anyone of the preceding claims wherein in a further step, a hybridisation assay is carried out and a hybridisation condition which is characteristic of the sequence is monitored.
A method for determining a characteristic of a sequence, said method comprising;
Dependencies are inferred from the claim text.