PROCESS FOR SYNTHESIZING COMPLETE-LENGTH cDNA, PROCESS FOR PRODUCING INTERMEDIATE THEREFOR, AND PROCESS FOR PRODUCING RECOMBINANT VECTOR CONTAINING COMPLETE-LENGTH cDNA

摘要

The invention discloses a method of providing a cDNA containing the whole of the information on the primary structure of a protein by selectively synthesizing only the complete-length cDNA that contains a sequence beginning with the cap site of a mRNA. The process for producing an intermediate for the synthesis of the complete-length cDNA comprises the step of treating a mRNA extracted from cells with an alkaline phosphatase to eliminate the phosphate group from the 5' end of an uncapped incomplete-length mRNA, the step of decapping from the 5' end of a capped complete-length mRNA, and the step of linking the 5'-end phosphate group formed in the above step to either the DNA oligonucleotide represented by the following general formula (I) or a DNA-RNA chimera oligonucleotide by means of a T4RNA ligase to selectively add the DNA oligonucleotide or DNA-RNA chimera oligonucleotide having an arbitrary sequence to the 5' end of the complete-length mRNA: 5'-dN1-dN2- (* f HANDWRITTEN *) -dNm-N1-N2- (* f HANDWRITTEN *) -Nn-3', wherein dN represents a deoxyribonucleotide selected from among dAMP, dCMP, dGMP and dTMP; N represents a ribonucleotide selected from among AMP, CMP, GMP and UMP; "-" represents a phosphoric ester linkage; m represents an integer of 1 or above; and n represents an integer of 0 or above. The process for synthesizing a complete-length cDNA comprises linking a double-stranded DNA primer having a dT tail by annealing to the poly (A) tail of the 3' end of the complete-length mRNA having the 5' end to which the DNA oligonucleotide or DNA-RNA chimera oligonucleotide have been added as described above, and then synthesizing a single-stranded cDNA complementary to the complete-length mRNA by means of a reverse transcriptase.