Seamless cloning and gene fusion

Abstract

Gene fusion technology is a key tool in facilitating gene function studies. Hybrid molecules in which all the components are joined precisely, without the presence of intervening and unwanted extraneous sequences, enable accurate studies of molecules and the characterization of individual components. This article reviews situations in which seamlessly fused genes and proteins are required or desired and describes molecular approaches that are available for generating these hybrid molecules.

Figure 1

Seamless gene fusion by overlap PCR. The diagram shows seamless fusion of DNA fragments X and Y. The two DNA fragments are PCR amplified individually. Primers P2 and P3 are designed so that the 5′-end 15 bases are complementary to each other. The PCR products are then used as templates for a second PCR amplification with primers P1 and P4. The complementary part of P2 and P3 could be part of fragment X or fragment Y. Note that to facilitate efficient PCR amplification, the melting temperature (T_m) for all primers should be made to be similar within the range of 55°C–75°C.

Figure 2

Seamless DNA manipulation by QuickChange™ site-directed mutagenesis. The diagram shows steps involved in site-directed mutagenesis for generating point mutations (a), insertions (b) or deletions (c). In all the cases, two complementary mutagenic primers (or megaprimers in case b) are used with each having >15 base homologous sequences flanking the mutagenic site. After primer extension cycles with Pfu polymerase, the undesired methylated template DNA and semi-methylated hybrids are fragmented by treating with restriction enzyme DpnI. The desired mutant circular duplexes are recovered in E. coli following transformation. The plasmid backbone contains an origin of replication (ori) and a selectable marker (sm).

Figure 3

Seamless gene cloning and gene fusion via a type IIS restriction enzyme. SapI is used as an example. (a) Seamless assembly of fragments X, Y, and Z. The fragments are first individually PCR amplified, with primers containing a SapI site. The primers are so designed that upon SapI digestion specific cohesive ends are generated for each fragment. Following SapI digestion, ligation of the fragments results in seamless assembly of X, Y, and Z. The 5′-end of X and the 3′-end of Z are made to contain SapI (with ends incompatible to other ends of X, Y and Z) or any other restriction sites for further subcloning. (b) Seamless fusion of fragment X with fragments Y and Z contained in a vector. Fragment A is PCR amplified with primers containing a SapI site. The primers are so designed that upon digestion with SapI cohesive ends are generated. The vector fragment, which contains fragments Y and Z, was specially engineered and prepared by SapI digestion. Ligation of the SapI-treated fragment A with the vector fragment results in seamless fusion of X with Y and Z. The plasmid backbone contains an origin of replication (ori) and a selectable marker (sm). Note that the nucleotide bases comprising the cohesive ends can be part of the fragment X or its fusion partners, and can be chosen to make ligation of the fragments directional.

Figure 4

Seamless cloning by ligation-independent cloning (LIC). The diagram shows seamless fusion of fragment X with fragments Y and Z contained in a vector. Fragment X is first PCR amplified and purified. The primers are designed so that the 5′-end 12 bases are free of one specific nucleotide (e.g. dT as an example). The product is then treated with a DNA polymerase possessing 3′-5′ exonuclease activity (such as T4 DNA polymerase and Pfu) in the presence of dATP. The polymerase starts to remove nucleotides from 3′-ends of the fragment until a dA base is encountered and removed, which is subsequently added back by the enzyme's 5′-3′ polymerase activity. This reaction generates 12 base (or longer) overhangs on fragment X. The vector fragment, which contains fragments Y and Z was engineered and prepared similarly to yield 12 base (or longer) overhangs that are complementary to the insert fragment. The plasmid backbone contains an origin of replication (ori) and a selectable marker (sm). The LIC-ready vector fragment and the insert fragment are then annealed to form circular duplexes, which are recovered in E. coli following transformation , . Seamless fusion of X with Y and Z is achieved if the nucleotides comprising the LIC overhangs are made to be parts of X or its fusion partners.

Figure 5

Seamless cloning by in vivo recombination. The diagram shows seamless fusion of fragment X with fragments Y and/or Z contained in a vector. Both the insert and the vector fragments are PCR amplified. The primers used in the reaction are designed so that the products contain a stretch of homologous sequences (15–40 bps) at the ends. The plasmid backbone contains an origin of replication (ori) and a selectable marker (sm). The two DNA fragments are then co-transformed into E. coli for in vivo recombination. Seamless fusion is achieved as long as the homologous sequences are parts of fragment X or its fusion partners.