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PMID: 8619474 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Analytical biochemistry ·Vol. 236 ·No. 1 ·1996-04-05 ·Pages 107-13

Andersson B, Wentland MA, Ricafrente JY, Liu W, Gibbs RA

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.

MeSH Terms

Base Sequence Cloning, Molecular/methods Genetic Vectors Genomic Library Humans Molecular Sequence Data Oligonucleotides/chemistry Polymerase Chain Reaction X Chromosome

Chemicals

Oligonucleotides

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Andersson B

Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

Wentland M A

Ricafrente J Y

Liu W

Gibbs R A

Article Info

Journal

Analytical biochemistry

Abbr.

Anal Biochem

ISSN

0003-2697

DOI

10.1006/abio.1996.0138

Published

1996-04-05

Pages

107-13

Language

English

Region

United States

NLM ID

0370535

Subset

Grants

NHGRI NIH HHS · R01 HG00823 · United States

Databases

GENBANK

AF007128, AF007129, AF007130, AF007131, AF007132, AF007133, AF007134, AF007135, AF007136, AF007137, AF007138, AF007139, AF007140, AF007141, AF007142, AF007143, AF007144, AF007145, AF007146, AF007147, AF007148, AF007149, AF007150, AF007151, AF007152, AF007153, AF007154

External Links

PubMed source DOI: 10.1006/abio.1996.0138 View journal details

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