| Autor: |
Andersson B; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA., Wentland MA, Ricafrente JY, Liu W, Gibbs RA |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical biochemistry [Anal Biochem] 1996 Apr 05; Vol. 236 (1), pp. 107-13. |
| DOI: |
10.1006/abio.1996.0138 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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