Nanoreporters and methods of manufacturing and use thereof

Abstract

The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled probes that are capable of binding to and identifying target molecules based on the probes' label codes. Methods of making and using such probes are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 753,758, filed on Dec. 23, 2005, and U.S. Provisional Application No. 843,528, filed on Sep. 8, 2006, the disclosures of each of which is incorporated herein by reference in their entireties.1. FIELD OF THE INVENTION[0002]The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled reporter molecules, referred to herein as labeled “nanoreporters,” that are capable of binding individual target molecules. Through the nanoreporters' label codes, the binding of the nanoreporters to target molecules results in the identification of the target molecules. Methods of making and using such nanoreporters are also provided. The nanoreporters can be used in diagnostic, prognostic, quality control and screening applications.2. BACKGROUND OF THE INVENTION[0003]This invention rel...

AI Technical Summary

Benefits of technology

[0011]The present invention relates to methods for the generation of a diverse population of uniquely-labeled molecules, preferably synthetic molecules, referred to herein as nanoreporters, that can be used for the detection, identification, and direct quantification of a wide variety of target molecules. The methods are advantageous in that they generate large numbers of distinctly labeled reporter molecules, each capable of detecting a single target molecule, starting from just a small number of different types of label monomers.

[0024]However, the second nucleic acid need not comprise a third label attachment region to which are attached (directly or indirectly) one or more label monomers that emit light constituting a third signal. Such probes are referred to herein as “ghost probes.” Ghost probes contain a target-specific sequence, which improves the hybridization kinetics of a nanoreporter to its target molecule, and, optionally, an affinity tag that can be used to immobilize and stretch the dual nanoreporter. In nanoreporter embodiments employing a ghost probe, the first molecule (to which the label monomers are attached and is sometimes referred to herein as a “reporter probe”) is preferably a nucleic acid molecule of about 2,000 to about 10,000 bases in length, more preferably about 5,000 to about 8,000 bases in length, and the second molecule (the “ghost probe”) is preferably a nucleic acid molecule of about 40 to about 250 bases in length, more preferably about 50 to 100 bases in length. In a specific embodiment, both the first molecule and the second molecule are DNA molecules. As used herein, the use of the terms “about” and “approximately” before a number or range means that number or range plus or minus 5%.

Problems solved by technology

Unfortunately, despite the miniaturization of array formats, this method still requires significant amounts of the biological sample.

However, in several cases, such as biopsies of diseased tissues or samples of a discrete cell type, the biological sample is in limited supply.

In addition, the kinetics of hybridization on the surface of a microarray is less efficient than hybridization in small amounts of aqueous solution.

Moreover, while methods exist to estimate the amount of nucleic acid present in a sample based on microarray hybridization result, microarray technology thus far does not allow for detection of target molecules on an individual level, nor are there microarray-based methods for directly quantifying the amount of target molecule in a given sample.

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