Patent ID: 6544736
Filing Date: 2003-04-08
Classification: C12N

Abstract:
A method for synthesizing cDNA including a 5-terminal sequence of a full-length mRNA with a cap structure from a mRNA sample containing the full-length mRNA with the cap structure and a non-full-length mRNA without a cap structure in a mixture, comprising:(a) removing the phosphate group at the 5â€²-terminus of the non-full-length mRNA in the mRNA sample; (b) removing the cap structure at the 5â€²-terminus of the full-length mRNA in the mRNA sample; (c) ligating an oligoribonucleotide of a predetermined sequence to the phosphate group at the 5â€²-terminus of the mRNA generated by step (b) in the sample, said oligoribonucleotide having a sequence prepared by generating a number of oligonucleotide sequences including various combinations of bases in a predetermined number, carrying out a homology search with a predetermined nucleotide sequence data base to determine the occurrence number of a sequence completely matching or differing by one base, and preparing a combination of plural sequences in a low-frequency occurrence group including a sequence at the lowest occurrence number; and (d) subjecting the mRNA ligated with the oligoribonucleotide at the phosphate group at the 5â€²-terminus to a reverse transcriptase process using as a primer a short-chain oligonucleotide capable of being annealed to an intermediate sequence within the mRNA, to synthesize a first-strand cDNA, wherein said oligoribonucleotide has a sequence selected from the group consisting of: 5â€²-GUUGCGUUAC-ACAGCGUAUG-AUGCGUAAGG-3â€² (SEQ ID NO;1), 5â€²-GUUGCGUUAC-ACAGCGUAUG-AUGCGUAA-3â€² (SEQ ID NO:2), 5â€²-GUUGCGUUAC-ACAGCGUAUG-AUGCGU-3â€² (SEQ ID NO:3), 5â€²-AAGGUACGCC-GUUGCGUUAC-ACAGCCUAUG-AUGCGU-3â€² (SEQ ID NO:4), 5â€²-AAGGUACGCC-GUUGCGUUAC-ACAGCGUAUG-AUGCGUAA-3â€² (SEQ ID NO:5), 5â€²-GUUGCGUUAC-AAGGUACGCC-ACAGCGUAUG-AUGCGU-3â€² (SEQ ID NO:6) and 5â€²-CUUGCGUUAC-AAGGUACGCC-ACAGCGUAUG-AUGCGUAA-3â€² (SEQ ID NO: 7).