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

US5597713A · kind A · utility

Assignees

• Kanagawa Academy of Science and Technology · JP
• Sagami Chemical Research Center

Inventors

• Seishi Kato · Sagamihara, JP
• Shingo Sekine · Sagamihara, JP

Key dates

• Filing date: Jul 25, 1994
• Grant date: Jan 28, 1997
• Expiry date: Jul 25, 2014

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/66
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

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. EQU 5'-dN1-dN2- . . . -dNm-N1-N2- . . . -Nn-3' [I] The 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 c…