Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations

A method for recognizing sequence identities and similarities between the sequence of 3′-ends of mRNA molecules present in a sample and a database of sequences, comprising the steps of:

• (a) preparing double-stranded cDNAs from a mRNA population using a mixture of 12 anchor primers, the anchor primers each including:
• (i) a tract of from 7 to 40 T residues;
• (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located to the 5′-side of the tract of T residues;
• (iii) a stuffer segment of from 4 to 40 nucleotides, the stuffer segment being located to the 5′-side of the site for cleavage by the first restriction endonuclease;
• and (iv) phasing residues -V-N located at the 3′-end of each of the anchor primers, wherein V is a deoxyribonucleotide selected from the group consisting A, C, and G;
• and N is a deoxyribonucleotide selected from the group consisting of A, C, G and T, the mixture including anchor primers containing all possibilities for V and N, to produce a cDNA sample;
• (b) producing cloned inserts from a suitable host cell that has been transformed by a vector, the vector having a double stranded cDNA insert that has been produced by the cleavage of the double stranded cDNA prepared in step (a) with a second restriction endonuclease and the first restriction endonuclease inserted in the vector, the cleaved double stranded cDNA being inserted in the vector in an orientation that is antisense with respect to a bacteriophage-specific promoter within the vector, the second restriction endonuclease recognizing a four-nucleotide sequence and the first restriction endonuclease cleaving at a single site within each member of the mixture of anchor primers;
• (c) generating linearized fragments of the cloned inserts by digestion with at least on restriction endonuclease that is different from the first and second restriction endonucleases;
• (d) generating cRNA preparation of antisense cRNA transcripts by incubation of the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter;
• (e) dividing the cRNA preparation into sixteen subpools and transcribing first-strand cDNA from each subpool, using a thermostable reverse transcriptase and one of sixteen primers, each having a 5′ and 3′-terminus, wherein said 3′-terminus is -N-N, wherein N is one of the four deoxyribonucleotides A, C, G, or T, and said 3′-terminus is complementary to the two nucleotides of a cRNA downstream from the second restriction endonuclease site located nearest the 3′ end of one strand of the cRNA sequence, the primer being at least 15 nucleotides in length, wherein said 5′-terminus is complementary to one strand of the vector sequence extending across the second restriction endonuclease site, and a different primer is used in each of the subpools;
• (f) using the first strand of cDNA produced by transcribing each of the sixteen subpools as a template for a polymerase chain reaction with a 3 ′-primer that is complementary to one strand of the vector adjoining the site of insertion of the cDNA sample in the vector and a 5′-primer selected grom the group consisting of:
• (i) the primer from which the first-strand cDNA was made for that subpool extended at its 3′-terminus by an additional residue -N, where N can be any of A, C, G, or T;
• (ii) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3′-terminus by two additional residues -N-N, wherein N can be any of A, C, G, or T, (iii) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3′terminus by three additional residues -N-N-N, wherein N can be any of A, C, G, or T;
• and (iv) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3′-terminus by four additional residues -N-N-N-N, wherein N can be any of A, C, G, or T, to produce polymerase chain reaction amplified fragments;
• and
• (g) resolving the polymerase chain reaction amplified fragments by electrophoresis to display bands representing the 3′-ends of mRNAs present in the sample;
• (h) eluting at least one cDNA corresponding to a mRNA from an electropherogram in which bands representing the 3′-ends of mRNAs present in the sample are displayed;
• (i) amplifying the eluted cDNA in a polymerase chain reaction;
• (j) cloning the amplified cDNA into a plasmid;
• (k) producing DNA corresponding to the cloned DNA from the plasmid;
• (l) determining the sequence of the cloned cDNA;
• (m) determining corresponding nucleotide sequences from a database of nucleotide sequences, said corresponding nucleotide sequences being delimited by the most distal recognition site for the second endonuclease and the beginning of the poly(A) tail;
• and comparing the sequence of the cloned cDNA to the corresponding nucleotide sequences, thereby recognizing sequence identities and similarities between the sequence of 3′-ends of mRNA molecules present in a sample and a database of sequences.