title: A "double adaptor" method for improved shotgun library construction.
abstract: The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.
authors.forename: B
authors.initials: B
authors.lastname: Andersson
authors.forename: M A
authors.initials: MA
authors.lastname: Wentland
authors.forename: J Y
authors.initials: JY
authors.lastname: Ricafrente
authors.forename: W
authors.initials: W
authors.lastname: Liu
authors.forename: R A
authors.initials: RA
authors.lastname: Gibbs
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D014960:X Chromosome
chemical_list: D009841:Oligonucleotides