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pubmed-article:8619474pubmed:abstractTextThe 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.lld:pubmed
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pubmed-article:8619474pubmed:authorpubmed-author:AnderssonBBlld:pubmed
pubmed-article:8619474pubmed:authorpubmed-author:LiuWWlld:pubmed
pubmed-article:8619474pubmed:authorpubmed-author:GibbsR ARAlld:pubmed
pubmed-article:8619474pubmed:authorpubmed-author:WentlandM AMAlld:pubmed
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pubmed-article:8619474pubmed:volume236lld:pubmed
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pubmed-article:8619474pubmed:authorsCompleteYlld:pubmed
pubmed-article:8619474pubmed:pagination107-13lld:pubmed
pubmed-article:8619474pubmed:dateRevised2007-11-14lld:pubmed
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pubmed-article:8619474pubmed:articleTitleA "double adaptor" method for improved shotgun library construction.lld:pubmed
pubmed-article:8619474pubmed:affiliationDepartment of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.lld:pubmed
pubmed-article:8619474pubmed:publicationTypeJournal Articlelld:pubmed
pubmed-article:8619474pubmed:publicationTypeResearch Support, U.S. Gov't, P.H.S.lld:pubmed
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