Title | A "double adaptor" method for improved shotgun library construction. |
Publication Type | Journal Article |
Year of Publication | 1996 |
Authors | Andersson, B, Wentland, MA, Ricafrente, JY, Liu, W, Gibbs, RA |
Journal | Anal Biochem |
Volume | 236 |
Issue | 1 |
Pagination | 107-13 |
Date Published | 1996 Apr 05 |
ISSN | 0003-2697 |
Keywords | Base Sequence, Cloning, Molecular, Genetic Vectors, Genomic Library, Humans, Molecular Sequence Data, Oligonucleotides, Polymerase Chain Reaction, X Chromosome |
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.
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DOI | 10.1006/abio.1996.0138 |
Alternate Journal | Anal Biochem |
PubMed ID | 8619474 |
Grant List | R01 HG00823 / HG / NHGRI NIH HHS / United States |