Use of mass labeled probes to detect target nucleic acids using mass spectrometry

Abstract

The invention relates to the use of mass labeled probes to characterise nucleic acids by mass spectrometry. Thus the invention provides methods of detecting the presence of a target nucleic acid in a sample, using a circularising probe in which a mass tag is present in the probe. Further methods of detecting the presence of a target nucleic acid are provided, which in contrast use a probe detection sequence in the circularising probe, wherein the probe detection sequence is detected with a probe attached to a mass tag. Methods for determining a genetic profile from the genome of an organism also form part of the invention.

Description

[0001]This application is a divisional of U.S. application Ser. No. 11 / 597,109, filed Nov. 20, 2006, which is a National Stage Application of PCT / GB2005 / 01980, filed May 19, 2004, which claims priority from U.S. Provisional Application 60 / 572,464, filed May 20, 2004, the entireties of which are hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING[0002]In accordance with 37 CFR §1.824, Applicant attaches herewith a copy of the Sequence Listing in computer readable form (CRF) in an electronic file, file name Sequence Listing.txt, created Nov. 20, 2006, file size 10.2 kilobytes, the contents thereof being incorporated by reference herein. The content of the sequence listing recorded in computer readable form is identical to the written sequence listing and, includes no new matter.FIELD OF THE INVENTION[0003]This invention relates to useful probe molecules for characterising biomolecules of interest, particularly nucleic acids. Specifically this invention relates to oligonucle...

AI Technical Summary

Benefits of technology

"The invention provides methods for detecting a target nucleic acid in a sample using a circularizing probe with a mass tag. The methods involve hybridizing a probe with the target nucleic acid, covalently connecting the ends of the probe to form a circularized-probe, cleaving the mass tag from the probe, and detecting the mass tag using mass spectrometry. The methods can be used in various applications such as genetic testing and disease diagnosis."

Problems solved by technology

However the costs of the detection apparatus and the difficulties of analysing the resultant signals limit the number of labels that can be used simultaneously in a fluorescence detection scheme.

However, multiplexed assays require more than just multiple tags.

Many nucleic acid probe binding assays do not function well when multiplexed because of problems of cross-hybridisation.

This is a particular problem for polymerase chain reaction (PCR) based assays, for which it is very costly and time-consuming to optimize reactions involving multiple primer pairs.

The problems are due to the high risk of cross hybridization of primers to incorrect templates leading to cross-amplification of templates and hence to incorrect results.

In addition, due to the substrate requirements of ligases, incorrectly hybridised probes with terminal mismatches at the ligation junction are ligated with very poor efficiency.

Similarly, mismatched probes, i.e. probes that have bound to a target that does not exactly match the probe sequence, are unable to ligate and therefore will not be circularized.

Despite the ability of mass tags to enable multiplexing of nucleic acid assays, none of the prior art on mass tags provides methods of analysing nucleic acids using circularising probes.

Similarly, none of the prior art on circularising probes provides methods of detecting circularising probes suggests using mass spectrometry.

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