Use of primer-restriction-end adapters in a novel cDNA cloning strategy.
We introduce a class of synthetic oligonucleotides, referred to as primer-restriction-end (PRE) adapters, which are bifunctional, one end serving as a primer for a polymerase reaction, while the other end can be ligated to restriction endonuclease digested DNA. Use of such adapters forms the basis of a new method for inserting single-stranded cDNA into cloning vectors, which involves very few separate biochemical modifications of the cDNA and so is appropriate when extensive fractionation of cDNA is desired prior to cloning. This novel methodology is highly efficient in producing full-length cDNA cloned in a predictable orientation within vectors, as we demonstrate by constructing and analysing clones of immunoglobulin lambda light chain cDNA in Escherichia coli.
Coleclough
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Erlitz
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D011088:DNA Ligases; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics
D004277:DNA, Single-Stranded; D007146:Immunoglobulin lambda-Chains; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D011088:DNA Ligases