A simplified and efficient vector-primer cDNA cloning system.
A simplified, efficient, and versatile vector-primer cDNA cloning system is presented. The dimer-primer system is a modification of the method of Okayama and Berg (1982) with the following features: (i) the vector-primer molecules are more rapidly and reliably prepared by virtue of the elimination of an endonuclease digestion and the agarose gel purification step from the original method, and (ii) the final cDNA products contain polylinkers at both cDNA-vector junctions, simplifying the size analysis, subcloning, and sequencing of inserts. The system is highly efficient, yielding greater than 10(5) transformants using 1 microgram mRNA and 1 pmol of vector-primer ends, with 75% or more of the transformants having inserts. The ability of the system to produce clones of full-length or near full-length is demonstrated by the analysis of 32 ribulose-1,5-bisphosphate carboxylase small subunit cDNA clones from tomato.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D010944:Plants; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D013698:Templates, Genetic; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D012333:RNA, Messenger; D005914:Globins