Patent ID: 6030784
Filing Date: 2000-02-29
Classification: C12N,C12Q

Abstract:
A method for simultaneous sequence-specific identification of mRNAs in a mRNA population 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 of 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 comprising the cDNA sample that has been cleaved with a second restriction endonuclease and the first restriction endonuclease and that has been 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 one restriction endonuclease that is different from the first and second restriction endonucleases;(d) generating a cRNA preparation of antisense cRNA transcripts by incubation of the linearized fragments with a bacteriophage-specific RNA polymerase that initiates 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 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 from 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 r, (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, thereby identifying, mRNAs in a mRNA population.