High-yield method for directional cDNA library construction.
Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.
Bellemare
G
G
Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada.
Potvin
C
C
Bergeron
D
D
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics*; D004256:DNA Polymerase I; D004274:DNA, Recombinant / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics*; D004722:Endoribonucleases; D015723:Gene Library; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D016914:Ribonuclease H
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes; D005914:Globins; D004247:DNA; D004256:DNA Polymerase I; D004722:Endoribonucleases; D016914:Ribonuclease H