Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms

Abstract

The invention comprises compositions and systems to identify and compare expressed genes in a given in vivo or in vitro RNA sample, as well as the relative difference in mRNA expression between two or more samples, where desired. Furthermore, the invention comprises compositions and systems to identify novel genes. The invention comprises, without limitation, one or more mRNA specific identimers for use in reverse transcription that themselves comprise an oligo-T nucleotide sequence (at the 5′ end) linked to a nucleotide sequence VNx (at the 3′ end) where the V nucleotide immediately adjacent to the oligo-T segment is not a T.

1 - 33 . (canceled)  
     
     
         34 . A system for identification and characterization of gene expression in one or more samples, comprised of: 
 (a) providing one or more samples comprising one or more mRNA molecules;    (b) providing an identimer comprising an oligo-dT primer of sequence, from 5′ to 3′ end, of Tn-VNx, where n is an integer 8 or greater but not more than 50 representing the number of T's , V equals a nucleotide A, C, or G but not T, each N equals a nucleotide A, C, G, or T, and x is an integer 3 or greater but not more than 10 representing the number of N nucleotides, said identimer also comprising a detectable marker at its 5′ end;    (c) contacting said mRNA with said identimer such that the polyT portion of the identimer hybridizes to the polyA tail of the mRNA and the VNx portion of the identimer hybridizes with portions of the mRNA immediately upstream of the polyA tail;    (d) reverse transcribing the mRNA to produce a first strand cDNA that includes the identimer;    (e) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex;    (f) cleaving the duplex with at least one sequence-specific cleaving agent to provide one or more duplex cleavage fragments;    (g) ligating an adaptamer comprising an RNA polymerase promoter site to one or more of said cleavage fragments; and    (h) amplifying the one or more ligated cleavage fragments using the identimer to produce one or more amplified fragments comprising sequences complementary to a 3′ end of the mRNA,    (i) identifying and characterizing the cleavage fragments according to the presence of the marker, the sequences corresponding to the VNx nucleotide sequence and the sequence associated with the sequence-specific cleaving agent, and the size of the fragment, and    (j) identifying any gene associated with the cleavage fragments by comparing the sequence and size characteristics of the cleavage fragment with a database contacting sequence and size characteristics of RNAs associated with known genes, whereby said comparison is conducted by means of software operated on a microprocessor,    where said detectable marker is comprised of a fluorescent molecule, a hapten, biotin, a radioisotope, or any combination thereof.