Process for producing cDNAs with complete length, process for producing intermediates thereof and process for producing vectors containing cDNAs with complete lengths

摘要

A process for providing cDNAs containing full primary structure information of proteins by selectively synthesizing full-length cDNAs containing sequences starting from the capped region of mRNAs is disclosed. The invention provides a process for producing an intermediate for the synthesis of a full-length cDNA, which comprises the steps of treating mRNA extracted from cells to eliminate the phosphate group from the 5' end of an uncapped degraded mRNA; decapping from the 5' end of a capped intact mRNA; and ligating either a DNA oligonucleotide or a DNA- RNA chimeric oligonucleotide represented by the following general formula [I] to the 5' end phosphate group formed in the above step by the action of T4 RNA ligase, thereby selectively adding either the DNA oligonucleotide or the DNA-RNA chimeric oligonucleotide having an arbitrary sequence to the 5' end of the intact mRNA.PP5'- dN1-dN2- . . . -dNm-N1-N2- . . . -Nn-3' [I]PPThe present invention also provides a process for synthesizing a full-length cDNA, which comprises linking a double-stranded DNA primer having a dT tail by annealing to a poly(A) tail of the 3' end of the intact mRNA having the DNA oligonucleotide or the DNA-RNA chimeric nucleotide added at the 5' end produced by the above-mentioned process, and then synthesizing the first strand cDNA complementary to the intact mRNA by a reverse transcriptase.