Amine Modified Random Primers for Microarray Detection
Description of Invention:
Available for licensing and commercial development is a new method for labeling nucleic acid molecules for use in hybridization reactions, and kits employing these methods. The fluorescence-labeled cDNA probes for DNA microarray studies only use about 1/20th as much input RNA as the conventional methods. The method allows making high quality probes from as little as 1 ug of total RNA without RNA or signal amplification. It is based on priming cDNA synthesis with random hexamers to the 5’ ends of which amino allyl modified bases have been added. Coupling of the fluorescent dye to the amine residues is performed after the cDNA is reverse transcribed. The method can be used in tandem with RNA amplification (and/or signal amplification) to label probes from 10 or fewer cells.
Furthermore, the invention also relates to a novel method to amplify RNA derived from single cells using T3-random 9mers and a new lysing method, which allow probe-labeling capabilities that are approaching the single cell level.
DNA Microarray technology has become one of the most important tools for high throughput studies in medical research with applications in the areas of gene discovery, gene expression and mapping. The suitability of DNA Microarray for profiling diseases and for identifying disease-related genes has also been also well documented. Most studies using DNA arrays involve preparation of fluorescent-labeled cDNA from the mRNA of the studied organism. The cDNA probes are then allowed to hybridize to the DNA fragments printed on the array, and the array is scanned and the data analyzed. Good results depend on a number of factors including high quality arrays and well-labeled probes. In order to achieve adequate sensitivity and reproducibility, probes have had to be prepared from rather large amounts of RNA using other methods.
Inventors:
Charles Xiang and Michael J. Brownstein (NIMH)
Patent Status:
DHHS Reference No. E-098-2001/0 --
U.S. Provisional Application No. 60/283,423 filed 11 Apr 2001
International Application PCT/US02/11656 filed 11 Apr 2002, which published as WO02083922 on 24 Oct 2002
U.S. Patent Application No. 10/474,611 filed 09 Oct 2003
and EP, CA and AU applications
Relevant Publication: Xiang CC, Kozhich OA, Chen M, Inman JM, Phan QN, Chen Y, Brownstein MJ. "Amine-modified random primers to label probes for DNA microarrays." Nat Biotechnol. 2002 Jul; 20(7): 738-42
Portfolios: Infectious Diseases Gene Based Therapies Devices/Instrumentation Cancer
Cancer -Research Materials-DNA Based Devices/Instrumentation-Diagnostics-Methods Infectious Diseases -Diagnostics-Bacterial Infectious Diseases -Diagnostics-Viral Cancer -Research Materials Devices/Instrumentation-Diagnostics Gene Based Therapies -Diagnostics Infectious Diseases -Diagnostics
For Additional Information Please Contact: Cristina Thalhammer-Reyero PhD MBA
NIH Office of Technology Transfer
6011 Executive Blvd, Suite 325
Rockville, MD 20852-3804
Phone: (301) 435-4507
Email: thalhamc@mail.nih.gov
Fax: (301) 402-0220
