University of California

John J Ngai

Tito A Serafini

David W Lin

Percy Luu

Ehud Y Isacoff

David M Lin

Lisa J Brunet

David J Speca

Jinhong Fan

Andrew H Dittman

The invention provides methods and compositions for normalizing and amplifying RNA populations. The methods generally comprise the steps of copying message RNA (mRNA) to form first single-stranded (ss) cDNA; converting the first ss-cDNA to first double-stranded (ds) cDNA; linearly amplifying the first ds-cDNA to form first amplified RNA (aRNA); tagging the 3' end of the first aRNA with a known sequence to form 3&#x2032;-tagged first aRNA; copying the 3&#x2032;-tagged first aRNA to form second ss-cDNA; and normalizing the mRNA and/or the first aRNA.

Normalizing and amplifying RNA

Nucleic acids are made by converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step contacting the single-stranded DNA with a DNA polymerase having 5&prime; exonuclease activity under conditions whereby the DNA polymerase converts the-single stranded DNA to the double-stranded DNA, wherein the single-stranded DNA is primed with oligonucleotide primer comprising a sequence complementary to the 3&prime; end of the single-stranded DNA, and at least one of the 5&prime; end of the primer and the single-stranded DNA comprises an RNA polymerase promoter joined to an upstream (5&prime;) flanking moiety which protects the promoter from the 5&prime; exonuclease activity of the DNA polymerase.

Methods for making nucleic acids

The invention provides methods and compositions for defining a cell type, generally involving the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type

Methods for defining cell types

The invention provides methods and compositions relating to odorant receptors, including a general expression cloning methodology which is useful for identifying novel G protein-coupled receptors and a novel family of odorant receptors and related nucleic acids, ligands, agonists and antagonists. These compositions provide a wide variety of applications such as screening for related receptors, and by modulating the function of the disclosed receptors by modulating their expression or contacting them with agonists, antagonist or ligands modulating reproductive/sexual and non-sexual social behaviors mediated via the olfactory system, reproductive physiologies and olfactory system regulated feeding behaviors, migratory behaviors and events such as conception, implantation, estrous and menstruation.

Odorant receptors

Nucleic acids are made by adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. In one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence, the cDNA is converted to double-stranded cDNA by a polymerase initiating from a noncovalently joined duplex region, and the double-stranded cDNA is transcribed to form one or more third RNAs.

The invention provides methods and compositions for defining a cell type, generally involving the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type.

Method of defining cell types by probing comprehensive expression

The invention provides methods and compositions for measuring the sense of smell, including animals having general anosmia as a result of a genetic mutation. The genetic mutation disrupts a function of a protein required for cyclic nucleotide mediated signal transduction in olfactory neurons in the animal. The invention also encompasses methods of making and using such animals, such as methods of characterizing the effect of a stimulus on the subject mutant animals by contacting the animal with a stimulus and measuring a response of the animal to the stimulus, wherein the presence of a response indicates that the stimulus evokes a non-olfactory response.

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Berkeley, CA, United States of America

John J Ngai