Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors.
We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.
Rutledge
R G
RG
Division of Biological Sciences, National Research Council of Canada, Ottawa.
Seligy
V L
VL
Côté
M J
MJ
Dimock
K
K
Lewin
L L
LL
Tenniswood
M P
MP
D001345:Autoradiography; D003001:Cloning, Molecular / Q000379:methods*; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D012313:RNA / Q000235:genetics*
D010761:Phosphorus Radioisotopes; D012313:RNA; D004247:DNA