Patent ID: 6596484
Filing Date: 2003-07-22
Classification: C12N,C12Q

Abstract:
A method for recognizing sequence identities and similarities between the sequence of 3â€²-ends of mRNA molecules present in a sample and a database of sequences, comprising the steps of:(a) preparing a double-stranded cDNA population from an mRNA population using a mixture of 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 5â€²-side of the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first stuffer segment being located to the 5â€²-side of the site for cleavage by the first restriction endonuclease; and (iv) phasing residues located at the 3â€² end of each of the anchor primers selected from the group consisting of -V and -V-N, 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 where the phasing residues in the mixture are defined by one of-V or -V-N; (b) cleaving the double-stranded cDNA population with the first restriction endonuclease and with a second restriction endonuclease, the second restriction endonuclease recognizing a four-nucleotide sequence, to form a population of double-stranded cDNA molecules having first and second termini, respectively; (c) inserting the double-stranded cDNA molecules from step (b) each into a vector in an orientation that is antisense with respect to a bacteriophage-specific promoter within the vector to form a population of vectors containing the inserted cDNA molecules, said inserting defining 3â€² and 5â€² flanking vector sequences such that 5â€² is upstream from the sense strand of the inserted cDNA and 3â€² is downstream of the sense strand, and said vector having a 3â€² flanking nucleotide sequence of from at least 15 nucleotides in length between said first restriction endonuclease site and a site defining transcription initiation in said promoter; (d) generating linearized fragments containing the inserted cDNA molecules by digestion of the vectors produced in step (c) with at least one restriction endonuclease that does not recognize sequences in the inserted cDNA molecules or in the bacteriophage-specific promoter, but does recognize sequences in the vector such that the resulting linearized fragments have a 5â€² flanking vector sequence of at least 15 nucleotides 5â€² to the site of insertion of the cDNA sample into the vector at the cDNA's second terminus; (e) generating a 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; (f) dividing the cRNA preparation into subpools and transcribing first-strand cDNA from each subpool, using a reverse transcriptase and a 5â€²-RT primer in each subpool, each one of the 5â€²-RT primers defined as having a 3â€²-terminus consisting of -Nx, wherein â€œNâ€  is one of the four deoxyribonucleotides A, C, G, or T, and â€œxâ€  is an integer from 1 to 5, the 5â€²-RT primer being 15 to 30 nucleotides in length and complementary to the 5â€² flanking vector sequence with the primer's complementarity extending across into the insert-specific nucleotides of the cRNA in a number of nucleotides equal to â€œxâ€ , wherein a different one of said 5â€²-RT primers is used in different subpools and wherein there are 4 subpools if â€œxâ€ =1, 16 subpools if â€œxâ€ =2, 64 subpools if â€œxâ€ =3,256 subpools if â€œxâ€ =4, and 1,024 subpools if â€œxâ€ =5; (g) using the product of first-strand cDNA transcription in each of the subpools as a template for a polymerase chain reaction with a 3â€²-PCR primer of 15 to 30 nucleotides in length that is complementary to 3â€² flanking vector sequences between said first restriction endonuclease site and the site defining transcription initiation by the bacteriophage-specific promoter and a 5â€²-PCR primer having a 3â€²-terminus consisting of Nxâˆ’-Ny, where â€œNâ€  and â€œxâ€  are as in step (f) -Nx is the same sequence as in the 5â€²-RT primer from which first-strand cDNA was made for that subpool, and â€œyâ€  is a whole integer such that x+y equals an integer selected from the group consisting of 3, 4, 5 and 6, the 5â€²-PCR primer being 15 to 30 nucleotides in length and complementary to the 5â€² flanking vector sequence with the 5â€²-PCR primer's complementarity extending across into the insert-specific nucleotides of the cRNA in a number of nucleotides equal to â€œx+yâ€ , to produce polymerase chain reaction amplified fragments; (h) resolving the polymerase chain reaction amplified fragments to generate a display of sequence-specific products representing the 3â€²-ends of different mRNAs present in the mRNA population; (i) eluting at least one of the sequence-specific products; (1) amplifying the eluted sequence-specific products in a polymerase chain reaction to produce amplified cDNA; (k) cloning the amplified cDNA into a plasmid; (l) producing DNA corresponding to the cloned DNA from the plasmid; (m) determining the sequence of the cloned DNA; (n) determining corresponding nucleotide sequences from a database of nucleotide sequences, said corresponding nucleotide sequences being delimited by the distal recognition site for the second endonuclease and the beginning of the poly(A) tail; and (o) comparing the sequence of the cloned cDNA to the corresponding nucleotide sequences, thereby recognizing sequence identities and similarities between the sequence of 3â€²-ends of mRNA molecules present in a sample and a database of sequences.