Clontech Laboratories, Inc.

Cloutech Laboratories, Inc.

Alex Chenchik

Paul Siebert

Luda Diatchenko

George G Jokhadze

Robert Bibilashvilli

Alexander Munishkin

Peter Simonenko

Eugene Sverdlov

Victor Tarabykin

Nadia Gurskaya

Konstantin Lukianov

Sergey Lukianov

York Zhu

Dmitry Bochkariov

Michael Herrler

Matvey Lukashev

Long oligonucleotide arrays, as well as methods for their preparation and use in hybridization assays, are provided. The subject arrays are characterized in that at least a portion of the probes of the array, and usually all of the probes of the array, are long oligonucleotides, e.g. oligonucleotides having a length of from about 50 to 120 nt. Each long oligonucleotide probe on the array is preferably chosen to exhibit substantially the same high target binding efficiency and substantially the same low non-specific binding under conditions in which the array is employed. The subject arrays find use in a number of different applications, e.g. differential gene expression analysis.

Long oligonucleotide arrays

Methods and compositions are provided for analyzing differences in the RNA profiles between a plurality of different physiological samples. In the subject methods, a set of a representational number of distinct gene specific primers is used to generate labeled nucleic acids from each of the different physiological samples. The labeled nucleic acids are then compared to each other and differences in the RNA profiles are determined. The subject methods find use in methods of identifying differential gene expression.

Methods of assaying differential expression

Arrays of a plurality of different heterogeneous polymeric target compositions immobilized on the surface of a solid support are provided. In the subject arrays, the constituent polymeric targets of the heterogeneous target compositions are generally biopolymeric compounds, e.g., nucleic acids and proteins, where ribonucleic acids and proteins are the preferred polymeric targets in many embodiments. The subject arrays find use in a variety of different applications, including high throughput gene expression analysis applications.

Polymeric arrays and methods for their use in binding assays

Methods and kits are provided for labeling nucleic acids, e.g. for use in array based hybridization assays. In the subject methods, target nucleic acid is generated from an initial nucleic acid source, e.g. mRNA, where the target nucleic acid is characterized by having at least one reactive functionality that is not a moiety found on naturally occurring nucleic acids. Functionalized label is then conjugated to the target nucleic acid, either before or after it has been hybridized to array of nucleic acids stably associated with the surface of a solid support. The subject methods find use in a variety of array based hybridization assays, including differential expression assays.

Methods of labeling nucleic acids for use in array based hybridization assays

Arrays of polymeric targets stably associated with the surface of a rigid solid support are provided. In the subject arrays, the polymeric targets are arranged at least according to size. The polymeric targets of the subject arrays are generally biopolymeric compounds, e.g. nucleic acids and proteins, where ribonucleic acids and proteins are the preferred polymeric targets. The subject arrays find use in a variety of different applications, and are particularly suited for use in high through gene expression analysis applications.

Mouse arrays and methods for their use are provided. The subject arrays include a plurality of polynucleotide spots, each of which is made up of a polynucleotide probe composition of unique polynucleotides corresponding to a key mouse gene. The subject arrays find use in hybridization assays, particularly in assays for the identification of differential gene expression of key mouse genes of interest.

Mouse arrays and kits comprising the same

The present invention pertains to methods for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5'-ends of mRNA molecules. The method of the present invention comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5'-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length ss cDNA includes the complete 5'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA. The subject invention also pertains to the template switching oligonucleotides that can be used according to the subject method. Kits containing the template switching oligonucleotide are also included within the scope of the present invention.

Methods and compositions for full-length cDNA Cloning using a

Described are compositions and methods which allow for the efficient addition of a defined sequence at the 3'-end of a full-length cDNA in the course of first-strand cDNA synthesis from an mRNA template. A cDNA synthesis primer that is capable of annealing to mRNA is used to prime the first strand synthesis reaction. An oligonucleotide that is linked to the 5'-end of the mRNA serves as a short, extended template such that when the reverse transcriptase enzyme reaches the 5'-end of the mRNA, the enzyme switches templates and proceeds to transcribe through the end of the linked oligonucleotide. As a result, the single-stranded cDNA product which corresponds to the full-length mRNA, will have at the 3'-end a defined sequence which is complementary to the linked oligonucleotide. A conservative element in the oligonucleotide sequence responsible for this reaction can include 3 to 5 guanylic acid residues at the 3'-end of the oligonucleotide. The subject invention provides for the increased synthesis of full-length cDNA from mRNA templates. The full-length cDNA prepared according to the present invention can then be amplified using PCR or cloned using standard procedures.

Methods and compositions for generating full-length cDNA having

The subject invention pertains to novel materials and methods for suppressing amplification of particular DNA fragments during polymerase chain reaction (PCR). The PCR suppression method uses novel adapters that are ligated to the end of a DNA fragment prior to PCR amplification. Upon melting and annealing, single-stranded DNA fragments having self-complementary adapters at the 5'- and 3'-ends of the strand can form suppressive 'pan-like' double-stranded structures that suppress amplification of the fragments during PCR. The subject method offers improved specificity and sensitivity of PCR amplification of a target DNA and does not require target DNA sequence information. The subject invention can be adapted to a variety of highly useful PCR techniques and applications.

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Palo Alto, CA, United States of America

Alex Chenchik