QUANTIFICATION OF NUCLEIC ACIDS AND PROTEINS USING OLIGONUCLEOTIDE MASS TAGS

摘要

The invention provides a method for detecting and quantifying the amount of target molecules, such as nucleic acids or proteins in a sample. The target molecules are first recognized and bounded by target-specific probes, generally nucleic acids or proteins that bind specifically to the targets, each of which is labeled with a short single-stranded nucleic acid probe, either DNA or RNA, with distinct molecular weight. This label is called an oligonucleotide mass tag. One or several standard oligonucleotide sequences can be designed with similar sequence but distinct molecular weight to those oligonucleotide mass tags. Then the oligonucleotide mass tags associated with bounded probes and the standard sequences are co-amplified using a pair of common primers. The presence and/or amount of each oligonucleotide mass tag, which corresponds to the amount of corresponding target molecule, is determined by a primer extension reaction and quantification of the primer extension product.

主权项

1. A method for detecting a target nucleic acid in a sample comprising the steps of
a) combining the target nucleic acid in a double-stranded form with a probe 1 and a probe 2, wherein
(i) the 5′ end of the probe 1 comprises a region A that is a nucleic acid sequence detected by a primer 1, a region B that comprises a first massTag sequence that has a known unique mass, and a region C that is the most 3′-region of the probe 1 and comprises a first target recognition sequence that binds the target, and(ii) the probe 2 comprises in its most 5′-region a region D that is a second target recognition sequence, followed by a region E that comprises a second massTag sequence, and a region F in its most 3′-region that is a constant region recognized by primer 2 andwherein the region C and the region D of the probes 1 and 2, respectively, anneal to the target; b) ligating the probe 1 and the probe 2 together, wherein the ligated product consists of a continuous nucleic acid sequence comprising 5′-A-B-C-D-E-F-3′; c) amplifying the 5 ‘-A-B-C-D-E-F-3’ ligated product using the primer 1 and primer 2; d) optionally removing excess dNTPs; and e) detecting the amplified nucleic acid.