A "double adaptor" method for improved shotgun library construction.
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.
Andersson
B
B
Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.
Wentland
M A
MA
Ricafrente
J Y
JY
Liu
W
W
Gibbs
R A
RA
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D014960:X Chromosome
D009841:Oligonucleotides