Method of assessing the amount of nucleic acid in a sample
OwnerJoakim Lundeberg, Mostafa Ronaghiinventors
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Abstract
The present invention provides a method of assessing the amount of target nucleic acid in a sample which comprises co-amplifying the target nucleic acid and at least one competitor nucleic acid molecule wherein each competitor molecule contains a unique discriminatory sequence, and determining the relative amounts of the respective amplicons, characterised in that determination is achieved by detecting a primer extension reaction using each said amplicon as template and a kit for use in such a method.
Claims27
A method of assessing the amount of target nucleic acid in a sample, which method comprises co-amplifying the target nucleic acid and at least one competitor nucleic acid molecule, wherein each competitor molecule contains a unique discriminatory sequence, said co-amplification yielding target- and competitor-derived amplicons respectively;
- performing a multiplicity of primer extension reactions using each said amplicon as template, wherein the various competitor- and target-based extension reactions are differentiated from one another;
- and determining the relative amounts of the respective amplicons, wherein said determination is achieved by detecting said primer extension reactions by luminometrically monitoring pyrophosphate release.
A method of assessing the amount of target nucleic acid in a sample, which comprises
- (i) co-amplifying the target nucleic acid and a known amount of a competitor nucleic acid molecule, wherein said competitor molecule comprises a unique discriminatory sequence, and is present in a known amount;
- (ii) performing multiple primer extension reactions on each respective amplicon, wherein each said primer extension reaction yields an extension product of different length;
- (iii) detecting the results of each said primer extension reaction, on each said respective amplicon;
- and
- (iv) comparing the said results to determine the relative amount of the respective amplicons in order to provide an assessment of the amount of target nucleic acid in the sample.
A method of assessing the amount of target nucleic acid in a sample, which comprises
- (i) co-amplifying the target nucleic acid together with multiple competitor nucleic acid molecules, wherein each said competitor molecule is different and comprises a unique discriminating sequence and wherein each said different competitor molecule is present in a different amount;
- (ii) performing a primer extension reaction using each said respective amplicon obtained in step (i) as template, using the same extension primer for each respective template, wherein said primer extension reaction is template-specific;
- and
- (iii) determining the amounts of the respective amplicons by selectively detecting the results of each said primer extension reaction, wherein each said primer extension reaction is differentiated from said other extension reactions;
- and
- (iv) assessing the amount of target nucleic acid in the sample from said amounts.
A method of assessing the amount of target nucleic acid in a sample, which comprises
- (i) co-amplifying the target nucleic acid together with multiple competitor nucleic acid molecules, wherein each said competitor molecule is different and comprises a unique discriminating sequence and wherein each said different competitor molecule is present in a different amount;
- (ii) performing a primer extension reaction using each said respective amplicon obtained in step (i) as template using a different extension primer for each said respective template, wherein each said extension primer contains a match at its 3′ end for the discriminatory sequence of one of said competitor molecules or for the equivalent region in said target molecule;
- (iii) determining the amounts of the respective amplicons by detecting the results of each said primer extension reaction;
- and
- (iv) assessing the amount of target nucleic acid in the sample from said amounts.
Dependencies are inferred from the claim text.