A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid.
A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs.
Spickofsky
N
N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Margolskee
R F
RF
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011061:Poly A / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011061:Poly A; D004247:DNA