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A "double adaptor" method for improved shotgun library construction.
Andersson, B; Wentland, M A; Ricafrente, J Y; Liu, W; Gibbs, R A.
Afiliação
  • Andersson B; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.
Anal Biochem ; 236(1): 107-13, 1996 Apr 05.
Article em En | MEDLINE | ID: mdl-8619474
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.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Biblioteca Genômica / Clonagem Molecular Limite: Humans Idioma: En Revista: Anal Biochem Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Estados Unidos
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Biblioteca Genômica / Clonagem Molecular Limite: Humans Idioma: En Revista: Anal Biochem Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Estados Unidos