Biomedical Engineering Reference
In-Depth Information
Chapter 16
Seamless Ligation Cloning Extract (SLiCE) Cloning Method
Yongwei Zhang , Uwe Werling , and Winfried Edelmann
Abstract
SLiCE (
S
eamless
Li
ligation
C
loning
E
xtract) is a novel cloning method that utilizes easy to generate
bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in
vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods,
facilitates seamless cloning by recombining short end homologies (15-52 bp) with or without fl anking
heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments
from bacterial artifi cial chromosomes or other sources. SLiCE is highly cost-effective and demonstrates the
versatility as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. We
established a DH10B-derived
E. coli
strain expressing an optimized
λ
prophage Red recombination system,
termed PPY, which facilitates SLiCE with very high effi ciencies.
Key words
SLiCE cloning, In vitro recombination, Seamless cloning, Bacterial cell extract,
DNA cloning, Recombinant DNA
1
Introduction
The generation of recombinant DNA molecules is an essential tool
in modern molecular biology. The conventional DNA cloning
strategies that have been used for several decades typically involve
the use of type II restriction enzymes to generate appropriate DNA
fragments, the modifi cation of DNA ends to generate blunt or
sticky ends, and the ligation of the DNA fragments to generate
plasmid or other type DNA vectors [
1
-
3
]. However, these proce-
dures depend on the presence of appropriate restriction sites to
generate both vector and insert molecules and often leave unwanted
sequences at the junction sites. In addition, the restriction enzymes
and modifying enzymes required for these manipulations are often
expensive making these procedures costly especially in high
throughput settings. To circumvent these limitations, we devel-
oped a new restriction site-independent cloning method that does
not leave any unwanted sequences at the junction sites (seamless)
and is based on in vitro recombination between short regions of
