Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.     

A bacteriophage lambda vector for cloning with BamHI and Sau3A

A phage lambda cloning vector has been constructed which contains a single site for the restriction endonuclease BamHI. Since Sau3A and BglII produce the same cohesive ends as BamHI, this vector can also be used to clone DNA fragments generated with either of these enzymes. We have used this vector to construct an Escherichia coli library using partial digestion with Sau3A. This vector will be most useful for applications requiring genetic analysis of cloned E. coli genes.     

A Simple CELI Endonuclease-Based Protocol for Genotyping both SNPs and InDels

Modern genetic analyses rely on efficient genotyping of single-nucleotide polymorphisms (SNP) or insertion/deletion length polymorphisms (InDel) in genomes. Methods available to genotype these polymorphisms include sequencing, cleaved amplified polymorphic sequence, high-resolution DNA melting, and microarray analyses, which are all rather tedious or expensive to set up for daily use. Here, we report a simplified label-free CELI endonuclease (CELI)-based protocol that enables us to detect both SNPs and InDels for fragment lengths between 500 and 6 kb. PCR-amplified target DNA fragments were annealed, cleaved by CELI, and analyzed either cost-effectively by agarose gel electrophoresis or automatically by capillary electrophoresis. The optimal amplification sizes, potential blind ends, and the maximum pooling capacities were examined for both electrophoresis protocols. We believe that the CELI-based genotyping protocol can be used in the detection of mutations, marker-assisted breeding, map-based cloning, and genome-wide association studies.     

LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme.

We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.     

Characterizing seamless ligation cloning extract for synthetic biological applications

Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects.     

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.     

A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles

Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.     

DelsGate, a robust and rapid gene deletion construction method.

With the increasing availability of fungal genome sequences there is great demand for fast, simple high-throughput methods to generate constructs for gene deletion. Here we describe a method that combines PCR and Gateway cloning technology together with use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. These constructs are then used to produce deletion mutants in the organism of interest following applicable methods for that species. In establishing this protocol we determined empirically that 1 kb was a suitable flank length to facilitate homologous recombination in our species of interest, Ustilago maydis. The method, which we have named DelsGate (Deletions via Gateway), consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for transformation of Ascomycetes and the Basidiomycete fungus U. maydis which causes corn smut disease. We have tested the reproducibility of the DelsGate approach by generating deletion constructs for 12 U. maydis genes. Although not tested here, the PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. DelsGate has the potential to be universal for all organisms with efficient transformation and homologous recombination systems.     

DelsGate, a robust and rapid gene deletion construction method

With the increasing availability of fungal genome sequences there is great demand for fast, simple high-throughput methods to generate constructs for gene deletion. Here we describe a method that combines PCR and Gateway cloning technology together with use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. These constructs are then used to produce deletion mutants in the organism of interest following applicable methods for that species. In establishing this protocol we determined empirically that 1 kb was a suitable flank length to facilitate homologous recombination in our species of interest, Ustilago maydis. The method, which we have named DelsGate (Deletions via Gateway), consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for transformation of Ascomycetes and the Basidiomycete fungus U. maydis which causes corn smut disease. We have tested the reproducibility of the DelsGate approach by generating deletion constructs for 12 U. maydis genes. Although not tested here, the PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. DelsGate has the potential to be universal for all organisms with efficient transformation and homologous recombination systems.     

DNA cloning without restriction enzyme and ligase

One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation.     

One-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system

Hairpin RNA-based RNA interference (hpRNAi) has become a powerful tool for exploring gene function in reverse genetics. Although, several methods are available for making constructs that express hpRNAi, multiple time-consuming cloning steps are usually involved. Here, we introduce an efficient and flexible hpRNAi vector construction method via the isothermal in vitro recombination system (IR-hpRNAi). For an IR-hpRNAi reaction, two PCR products of a target gene sequence are generated, which containS complementary ends (~20 bp) to each other and to the ends of linearized vector, are fused in a way of head-to-head or tail-to-tail into the vector. This IR-hpRNAi method offers two options to construct the RNAi vectors. Using this method, we created a IR-hpRNAi construct for the Arabidopsis PDS3 gene,and verified the silencing effect via Agrobacterium-mediated transformation. The IR-hpRNAi system rules out the requirement of engineering restriction enzyme cutting sites in target DNA fragments, and is ligation-independent. Thus, this method has advantages over the other hpRNAi construction methods.     

Ligation-independent cloning of PCR products (LIC-PCR).

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.     

Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda

The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.     

Efficient ligation and cloning of DNA fragments with 2-bp overhangs

Various methods of ligation are currently available and routinely used by molecular biologists, such as blunt end ligation, cohesive end (two and four overhangs), and ligation of Taq polymerase-derived products. However, there is no efficient method for the cloning of DNA fragments with 2-bp overhangs. We present a simple method for the efficient ligation of DNA fragments with 2-bp overhanging ends, ranging in size from 0.7 to 2.5 kbp. Our method involves the initial heating and flash freezing of the vector-insert DNA mix, and a subsequent unique ligation reaction. This method provides a new molecular biology tool for researchers.     

Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway^TM cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway^TM cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.     

A ligation-independent cloning method using nicking DNA endonuclease

Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.     

A Rapid and Simple Method for DNA Engineering Using Cycled Ligation Assembly

DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method’s utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.     

Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.     

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was "blunt-end ligation" subsequent to "fill-in" or "blunting", with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.