Direct
Cloning of the Unknown Flanking DNA Fragmentsfrom a Large Insert without
Restriction Mapping
SONG Bao-Liang1, QI Wei1，2,LI Bo-Liang1*
( 1State Key Laboratory of Molecular Biology, Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the
Chinese Academy of Sciences, Shanghai 200031,China; 2
Department of Biological Science and Technology, Nanjing University, Nanjing 210093,
China )
Abstract A simple
technique for direct cloning of the target DNA fragments from a large insert
according to its adjacent known sequence is described here.In this new
subcloning method,a large DNA insert is digested and ligated with a linearized
plasmid vector to construct a subclone library that is subjected to
screening.The bacterial clones in this library are individually picked, grown
in a 96-well plate,and then pooled across the rows or columns. Target clones
are obtained from the ordered separate pools by PCR-screening with a set of
primers,one specific for the adjacent known sequence and the other serving as
“anchor primer” specific for the vector sequence.This direct subcloning
procedure was efficiently
demonstrated by cloning a specific DNA region from a large insert within 2 days
without mapping the starting DNA or isolating the digested DNA fragment.
Key words subclone;
screening; walking; ACAT
*Corresponding author: Tel,
86-21-64747035; Fax, 86-21-64338357; e-mail, [email protected]