Biomedical Engineering Reference
In-Depth Information
Chapter 5
USER-Derived Cloning Methods and Their Primer Design
Bo Salomonsen , Uffe H. Mortensen , and Barbara A. Halkier
Abstract
Uracil excision-based cloning through USER™ (Uracil-Specifi c Excision Reagent) is an effi cient ligase-free
cloning technique that comprises USER cloning, USER fusion, and USER cassette-free (UCF) USER
fusion. These USER-derived cloning techniques enable seamless assembly of multiple DNA fragments in
one construct. Though governed by a few simple rules primer design for USER-based fusion of PCR frag-
ments can prove time-consuming for inexperienced users. The Primer Help for USER (PHUSER) soft-
ware is an easy-to-use primer design tool for USER-based methods. In this chapter, we present a PHUSER
software protocol for designing primers for USER-derived cloning techniques.
Key words
Primer design, USER fusion, PCR-based cloning, Seamless DNA fusion, Ligation-free
cloning, PHUSER
1
Introduction
The invention of tools allowing DNA fragments to be combined in
vitro has served as a cornerstone in both molecular biology and
biotechnology. The original technology, discovered by Mertz and
Davis in 1972 [
1
], combines DNA ligation of any two comple-
mentary cohesive ends [
2
,
3
] formed through digestion with
restriction endonucleases [
4
]. Since then other discoveries have
made cloning a routine operation within molecular biology. Most
noteworthy was the implementation of strategies based on the
polymerase chain reaction (PCR) [
5
,
6
] that allows for incorpora-
tion of restriction enzyme recognition sites in the primers for direct
cloning of any DNA fragment [
7
]. Moreover, PCR gave rise to a
plethora of new methods for DNA manipulation including, e.g.,
site-directed mutagenesis and assembly of multiple DNA frag-
ments by overlap extension [
8
-
11
]. Importantly, PCR released
cloning from the limitations of the traditional restriction digest
and ligation cloning method [
12
-
17
] as it enabled introduction of
complementary bases in the ends of one or more linear DNA frag-
ments. These ends can then be joined by creating single-stranded
