A "double adaptor" method for improved shotgun library construction.

TitleA "double adaptor" method for improved shotgun library construction.
Publication TypeJournal Article
Year of Publication1996
AuthorsAndersson, B, Wentland, MA, Ricafrente, JY, Liu, W, Gibbs, RA
JournalAnal Biochem
Date Published1996 Apr 05
KeywordsBase Sequence, Cloning, Molecular, Genetic Vectors, Genomic Library, Humans, Molecular Sequence Data, Oligonucleotides, Polymerase Chain Reaction, X Chromosome

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.

Alternate JournalAnal Biochem
PubMed ID8619474
Grant ListR01 HG00823 / HG / NHGRI NIH HHS / United States

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