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US 2003/0148525 A1Published application
Isometric primer extension method and kit for detection and quantification of specific nucleic acid
OwnerXiao Bing Wanginventor
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Abstract
A method for detecting and/or quantifying a target DNA or RNA present in a sample by a isometric primer extension method is disclosed. The method includes carrying out a primer extension reaction in the absence of a free nucleotide so that the primer extension reaction is stopped where the absent nucleotide would have been inserted. Thus, as the amount of incorporation of a labeled nucleotide on the primer extended product is detected, the amount of the target RNA or DNA in the sample is measured.
Claims40
A method for detecting or quantifying a target nucleic acid in a sample comprising:
- (a) preparing a primer or primers specifically matched to a predetermined position of the target nucleic acid;
- (b) annealing the primer or primers from (a) with the target nucleic acid under high stringency conditions to obtain a primer-nucleic acid duplex at the predetermined position of the target nucleic acid;
- (c) mixing the primer-nucleic acid duplex from (b) with a mixture comprising:
- (1) one or two or three types of free non-terminator nucleotides and at least one type of non-terminator nucleotide that is optionally labeled with a detectable marker, and
- (2) with or without a type of terminator nucleotide that is different from the one or two or three types of non-terminator nucleotides in (1);
- (d) performing the primer extension by enzymatic or chemical reaction in an appropriate buffer;
- and
- (e) detecting or quantifying the amount of labeling signal on the primer extended nucleotides, or
- (f) detecting or quantifying the amount of extended primers by mass spectrometry.
6 more elements
Dependencies are inferred from the claim text.