title: Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers.
abstract: Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5'→3' exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes.
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore 138648, Singapore. shanshan_howland@immunol.a-star.edu.sg
authors.forename: Shanshan W
authors.initials: SW
authors.lastname: Howland
authors.forename: Chek-Meng
authors.initials: CM
authors.lastname: Poh
authors.forename: Laurent
authors.initials: L
authors.lastname: Rénia
mesh_terms: D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics*; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D010710:Phosphates / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010710:Phosphates; C083120:phosphorodithioic acid; D004262:DNA Restriction Enzymes