Description of Invention:
Available for licensing and commercial development are methods of labeling nucleic acid probes for the detection of nucleic acids molecules, for instance producing labeled probes for detecting hybridization signals, such as those from a microarray. This disclosure provides new methods for amplifying nucleic acid templates from very small samples, even as small as one cell. Nucleic acid templates amplified by the disclosed methods can be used in combination with any method that requires amplified nucleic acid. In addition, the amplified nucleic acid can be labeled with any labeling method, such as the labeling method disclosed herein. Also provided are methods for preparing modified nucleotide probes, from either amplified or unamplified nucleic acid templates. In one embodiment, the method includes the incorporation of modified nucleic acids into random primers that are used to initiate polymerization of a probe molecule. In another embodiment, the random primers include nucleotides that are modified by amine groups (such as aminoallyl moieties). In yet other embodiments, the modified nucleotides comprise a detectable molecule, such as a fluorophore or hapten. The disclosure also provides an improved method of extracting RNA from fixed cells or tissue sections for subsequent use as RNA templates or for generating labeled probe. In one specific embodiment, the cells are fixed with Dithio-bis(Succinimidyl Propionate) (DSP). Also disclosed are kits for producing a labeled hybridization probe, using a modified random primer, or for probing an array, and kits for amplifying nucleic acid templates from very small samples.
Inventors:
Charles Xiang and Michael J. Brownstein (NIMH)
Patent Status:
DHHS Reference No. E-098-2001/1 --
U.S. Patent Application No. 10/269,515 filed 11 Oct 2002, published as US2003170675 on 11 Sept 2003
DHHS Reference No. E-098-2001/2 --
International Application PCT/US03/33319 filed 10 Oct 2003, published as WO 200/033669 on 22 April 2004
U.S. Patent Application No. 11/104,737 filed 11 Apr 2005
Relevant Publication:
Xiang CC, Chen M, Kozhich OA, Phan QN, Inman JM, Chen Y, Brownstein MJ. “Probe generation directly from small numbers of cells for DNA microarray studies.” Biotechniques. 2003 Feb;34(2):386-8, 390, 392-3
Xiang CC, Chen M, Ma L, Phan QN, Inman JM, Kozhich OA, Brownstein MJ. “A new strategy to amplify degraded RNA from small tissue samples for microarray studies.” Nucleic Acids Res. 2003 May 1;31(9):e53
Xiang CC, Brownstein MJ. “Preparing fluorescent probes for microarray studies.” Methods Mol Biol. 2003; 224:55-60
Xiang CC, Mezey E, Chen M, Key S, Ma L, Brownstein MJ. “Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling” Nucleic Acids Res. 2004 Dec 16;32(22): e185
Portfolios: Infectious Diseases Gene Based Therapies Devices/Instrumentation Cancer
Cancer -Diagnostics-In Vitro-DNA Based Cancer -Diagnostics Cancer -Research Materials Devices/Instrumentation-Diagnostics Devices/Instrumentation-Research Materials Gene Based Therapies -Diagnostics Gene Based Therapies -Research Materials 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
