The development of a novel vector for construction of a full-length cDNA library

Abstract

A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.