A Guide to Cloning the Products of Polymerase Chain Reactions

BACKGROUND

Since the first cloning papers were published in the early 1970s, hundreds of different methods have been published describing how best to generate and assemble DNA constructs of increasing complexity and sophistication. In the days before PCR, these methods—of necessity—were permutations and modifications of just two enzymatic reactions: restriction and ligation. Despite the inventiveness of the early researchers, precise assembly of complex DNA structures remained a significant challenge. Hardly surprising then, that only one or two of the cloning methods that were so carefully developed in those early days remain in common use today. Most were simply swept aside in the late 1980s by the liberating tide of PCR. During the course of 25 years, an expanding repertoire of PCR techniques has come to dominate molecular cloning.

The following guide outlines various methods used to clone amplified DNAs and to facilitate the construction of complex multicomponent genetic units (for an overview of standard methods, see Table 1). [Note that the names and products of the manufacturers mentioned below were current at the time of this initial writing.]

SITE-SPECIFIC RECOMBINATION METHODS

In the last few years, cloning and subcloning of PCR products and other DNAs into prokaryotic vectors has undergone a revolution. No longer is it necessary to depend on ligation of compatible termini or on complex PCR protocols. Instead, DNA segments flanked by the appropriate recombination sites can be joined in vitro to vectors carrying compatible recombination sites through the action of sequence-specific recombinases such as bacteriophage λ integrase.

Recombinational cloning has several advantages over more traditional methods of cloning that rely on the restriction enzymes and ligases. Recombinational cloning:

• is flexible and highly efficient

• enables DNA fragments to be moved easily and rapidly from one expression vector to another in a single-step procedure that maintains orientation and reading frame

• facilitates protein expression and functional analysis

• can be adapted to high-throughput, automated formats

• has produced large archives of clones, carrying mammalian open reading frames generated by the Gateway cloning system, that are publicly available from the Mammalian Gene Collection at NIH (http://mgc.nci.nih.gov/)

Originally developed for large-scale proteomic analyses, recombinational cloning is increasingly used for small-scale research projects and for most large-scale proteomic analyses. The two most popular versions of recombinational cloning—Gateway cloning (Life Technologies) and In-Fusion (Clontech)—use different sequence-specific recombinases and have different strengths and weaknesses. Both systems are patent protected.

1. Gateway cloning (Life Technologies) uses an in vitro version of the site-specific recombinational system used by bacteriophage λ DNA during integration into and excision from the E. coli genome (Hartley et al. 2000; Walhout et al. 2000; see also Introduction: Gateway Recombinational Cloning [Reece-Hoyes and Walhout 2018]). Three enzymes are involved: integrase (Int), excisionase (Xis), and integration host factor (IHF). These enzymes catalyze recombination at specific DNA sequences known genetically as attB, attP, attL, and attR. The Gateway system uses modified versions of these sequences and their cognate recombinases that increase the specificity of the recombination reaction without reducing its efficiency (see Table 2).

   In Gateway cloning, a DNA fragment carrying attB1 at one end and attB2 at the other is prepared by PCR and cloned into a vector (the donor vector) carrying attP1 and attP2 recombination sites flanking the ccdB gene, a death gene in E. coli (for information about the ccdB gene and its protein, see Box 1). Cloning is performed in an in vitro recombination reaction, driven by a proprietary enzyme (BP clonase), during which the incoming DNA fragment replaces the ccdB sequences. The resulting recombinant plasmid is called an Entry clone and its flanking recombination sequences are called Gateway attL-type.

   The DNA sequence carried by the Entry clone can then be transferred into any Destination vector that carries attR recombination sequences—a reaction that is catalyzed by LR Clonase—a proprietary mixture of enzymes. The power of the Gateway system is that it allows DNA fragments flanked by appropriate sequence-specific recombination sites to be transferred into a Destination vector in a highly efficient single-step procedure. Thousands of Destination vectors are available, equipped with prokaryotic, mammalian and viral promoters, signal sequences, splicing signals, Shine–Dalgarno sequences, cleavage sequences, poly(A) addition sequences, and many other elements required for (i) the expression in various types of cells and (ii) purification of the target protein. For further details on Gateway cloning, see Introduction: Gateway Recombinational Cloning (Reece-Hoyes and Walhout 2018) and Figures 1, 2, and 3 therein.

2. In-Fusion cloning uses a proprietary version of vaccinia virus-encoded DNA polymerase called the In-Fusion enzyme. This enzyme has three catalytic functions—strand displacement, exonucleolytic removal of nucleotides, and homologous recombination (Marsischky and LaBaer 2004; Hamilton et al. 2007). In reactions containing two double-stranded linear DNA substrates the 3′–5′ proofreading activity of the poxvirus DNA polymerase removes nucleotides from the 3′ termini of the DNAs. If the exposed single-stranded regions of the two molecules are homologous in sequence, they anneal to form a best-fit hybrid that is relatively resistant to further exonucleolytic attack by the polymerase. Any nicks, short overhangs, and gaps are repaired after the annealed DNAs are introduced into E. coli, using a standard transformation protocol.

   In the In-Fusion system, the regions of homology are generated by adding to the forward and reverse PCR primers 15-base extensions that exactly match the known sequences at the ends of a linearized plasmid vector. The In-Fusion system can therefore be used to create circular recombinant plasmids from any DNA fragment whose terminal sequences have been appropriately tailored by PCR (Benoit et al. 2006; Zhu et al. 2007).

   Clontech markets plasmid vectors that can be used to create Master clones in which the segment of cloned DNA is flanked by loxP sites (see Fig. 1). The sequences inserted into a Master clone can then be to any Acceptor vector that also carries a loxP site in an in vitro reaction catalyzed by Cre recombinase. A collection of Acceptor vectors engineered to express proteins in bacteria, yeast, and mammalian cells is available from Clontech. Like Gateway's Destination vectors, In-Fusion Expression vectors are equipped with a variety of signals, tags, and selective markers.

   In summary, the In-Fusion system is simple, efficient, and rapid and can be adapted for high-throughput projects (e.g., the BioBricks project).

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Figure 1.

Ligation-independent cloning using In-Fusion (Clontech). (A) The reaction mixture contains a vector of choice linearized by a restriction cut, a PCR product generated with primers containing 15-bp 5′ ends homologous to the ends of the linear vector (gene), and the proprietary In-Fusion enzyme. (B) The enzyme catalyzes the alignment and strand displacement of the homologous ends of the PCR product with the vector, whereas 3′-exonuclease activity removes single-stranded regions. (C) The nicks are sealed after transformation of E. coli. (Reproduced from D'Arpa 2009.)

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Figure 2.

TOPO tools (P_CMV/TetO 5′ element) for producing linear DNA constructs with defined 5′ and 3′ functional elements. (A) The 5′ and 3′ functional elements and the PCR product to which they are to be added are shown. Different 11-bp sequences (bold) are added to the ends of the PCR product via forward and reverse primers. (B) TOPO cleaves the PCR product, creating TOPO-activated overhangs (and the release of single-stranded 6-mers) complementary to the overhangs of the TOPO-activated functional elements. (C) Annealing the overhangs juxtaposes the 5′-OH of each to attack the phosphotyrosyl bond of the other, ligating both strands of each functional element to both strands of the PCR product, releasing two TOPO molecules, and creating a linear recombinant template for direct use in vitro and in vivo. (Reproduced from Invitrogen 2002, part of Life Technologies, 2002.)

OTHER CLONING METHODS

TOPO TOOLS: CREATING LINEAR EXPRESSION CONSTRUCTS WITH FUNCTIONAL ELEMENTS

Chimeric DNA constructs remain the workhorses of much of molecular biology. Expression constructs typically consist of a series of functional units (promoters, open reading frames, signals for directing the traffic of protein to particular cellular locations, reporters, immunological tags, and terminators). Traditionally, these elements were joined together one at a time—often painfully slowly—by a combination of restriction enzyme digestion, ligation, and, when necessary, site-directed mutagenesis. The publication of the first paper outlining a PCR-based methodology for assembly and use of linear expression vectors (Sykes and Johnston 1999) opened the way to the development of functional DNA elements linked to vaccinia virus topoisomerase (TOPO Tools, Life Technologies; see Table 3). In this elegant system, the coding sequences of interest are first amplified using primers that introduce a vaccinia virus topoisomerase recognition site and ancillary sequences upstream and downstream of the coding sequence (see below). The A-tailed PCR products are then mixed with TOPO Tools elements, which are functional nucleic acid sequences (promoters, fusion tags, termination sequences, etc.) bound to topoisomerase, which catalyzes joining of the functional elements to the PCR products in a rapid (5–10 min) reaction at room temperature. After the desired 5′ or 3′ elements have been added, the resulting recombinants are amplified in a second PCR to generate functional linear cassettes that can be directly used in downstream applications (see Fig. 2). The technology can easily be adapted for use in 96-well or 393-well formats.

The manual on TOPO Tools Technology (Invitrogen 2002) contains clear, detailed descriptions of all of the steps and the necessary controls required to link user-generated PCR products to the 5′- and 3′-TOPO-adapted elements sold by Life Technologies. This information can be outlined as follows.

• 1. Design PCR primers to amplify the target sequence. These primers carry 5′-HO groups and should contain the 11-bp sequences required for joining the PCR product to TOPO Tools 5′ and 3′ elements.

• 2. Use a high-fidelity Taq polymerase (e.g., Platinum Taq, Life Technologies) to amplify the target sequence and, using gel electrophoresis, verify the integrity and concentration of the PCR product.

• 3. Mix the PCR product in the appropriate molar ratios with the 5′- or 3′-TOPO-adapted elements to create a linear construct.

• 4. Use element specific primers (supplied by Life Technologies) in a second round of PCR to amplify the target construct and its attached elements. Verify the integrity and concentration of the amplified product, which if everything has gone well, should be ready for transfection or other downstream applications.