Type II Restriction Enzymes Research Articles

A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences

Ligation-mediated polymerase chain reaction (LM-PCR) is a whole genome amplification (WGA) method, for which genomic DNA is cleaved into numerous fragments and then all of the fragments are amplified by PCR after attaching a universal end sequence. However, the self-ligation of these fragments could happen and may cause biased amplification and restriction of its application. To decrease the self-ligation probability, here we use type IIS restriction enzymes to digest genomic DNA into fragments with 4–5nt long overhangs with random sequences. After ligation to an adapter with random end sequences to above fragments, PCR is carried out and almost all present DNA sequences are amplified. In this study, whole genome of Vibrio parahaemolyticus was amplified and the amplification efficiency was evaluated by quantitative PCR. The results suggested that our approach could provide sufficient genomic DNA with good quality to meet requirements of various genetic analyses.

A novel approach to the generation of seamless constructs for plant transformation.

BackgroundWhen creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors. Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition sequences from the vector leaving no undesired, short sequences behind.ResultsWe designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent protein, gfp, was successfully cloned into both vectors; traces of pAUrumIII, however, contaminated the transformation due to incomplete linearization, an issue not encountered with the type IIS linearized pAUrumII. Both constructs, pAUrumII-gfp and pAUrumIII-gfp, were functional, when tested in vitro on wheat and barley endosperm cells for transient gfp expression.ConclusionsAll nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we believe that the SRL system can be useful in a series of plant transformation vectors, favoring the presence of functional sequences for optimal expression over redundant cloning-site remnants.

Restriction Enzyme Body Doubles and PCR Cloning: On the General Use of Type IIS Restriction Enzymes for Cloning

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Transposon-based approaches for generating novel molecular diversity during directed evolution.

This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restriction endonuclease generates blunt-end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.

GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.

Establishing a versatile Golden Gate cloning system for genetic engineering in fungi

The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules allow not only simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.

A sequence-specific nicking endonuclease from streptomyces: purification, physical and catalytic properties.

A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease.

Rapid Cloning For Protein Crystallography Using Type IIS Restriction Enzymes

Recombinant proteins are used routinely in macromolecular crystallographic experiments. Efficiency in cloning thus becomes a valuable resource to a macromolecular crystallographer. The use of type IIS restriction enzymes (outside cutters) concurrent with ligation allows for rapid cloning. A vector can be modified easily to incorporate sites for outside cutters, such as BspQI or BsaI. Critical and unique to our cloning method is the upshift of reaction incubations to 50 °C where the ligase is inactivated while the restriction enzyme is still active. The result is that very low background of undesired cloning intermediates is observed. Multiple DNA molecules can be simultaneously joined with a simplicity and effectiveness that rivals overlap PCR when BsaI is used. Using type IIS enzymes thus provides great control, flexibility, and simplicity in cloning strategies.

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

UPDATE ON 'MOLECULAR SCISSORS' FOR TRANSGENIC FARM ANIMAL PRODUCTION

Molecular scissors, such as meganucleases, zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN), are valuable tools for generating double-strand breaks (DSB) in the genome that can lead to a functional knockout of the targeted gene or used to integrate a DNA sequence at a specific locus in the genome. Especially in farm animal species from which true pluripotent embryonic stem cells have not been established, these molecular scissors are a new option for engineering the genome in a way that was not feasible before. Meganucleases (also called homing nucleases) are natural proteins found in many single-cell organisms that are mainly involved in the cell’s repair mechanism after a strand break occurs. They are capable of recognising their binding site by identifying a sequence containing between 12 and >30 base pairs. The prototype enzyme for demonstrating DSB stimulation of gene targeting was I-SceI, which has a long recognition site (I-SceI 18 bp). The recognition specificity of enzymes such as I-SceI can be modified to be specific for a desired sequence within the genome. The use of meganucleases to genetically modify organisms has proved very successful in several species, including frog, fly, fish, plants, and human cells, but the intimate connection between the recognition and cleavage elements in the protein structure makes it difficult to alter one without affecting the other. The class of targeting reagents that has proved the most versatile and effective in recent years is that of ZFN. The ZFN possess separate DNA-binding and cleavage domains, which facilitate design according to the desired target. These molecules originate from the natural type IIS restriction enzyme FokI (Li et al. 1992 Proc. Natl. Acad. Sci. USA 89, 4275–4279). The cleavage domain has no sequence specificity and the binding domain can be used to make ZFN specific to a targeted sequence. The requirement for dimerisation of the FokI makes ZFN even more specific and avoids off-target events, as a monomeric cleavage does not occur at single binding sites. One zinc-finger molecule is specific for a base triplet; joining several zinc-finger molecules is sufficient to pick out a single target in a complex genome. ZFN have been used to modify the genome of several species as Xenopus, drosophila, C. elegans, zebrafish, rat, mouse, human cells, hamster cells, rabbit, pigs, and cattle. Different methods have been used to alter the host genomes either by ZFN mRNA or DNA injection into zygotes or by transfection of somatic cells followed by somatic cell nuclear transfer. Even a direct delivery of ZFN proteins can generate a targeted mutation (Gaj et al. 2012 Nat. Methods 9, 805–807). The efficiency of ZFN-mediated knockout was increased up to 10,000-fold compared with traditional gene knockout by homologous recombination. Rarely, off-target events were described but most were located in an intergenic or intronic region of the genome. Transcription activator-like effectors are a family of virulence factors produced by a genus of plant pathogens, Xanthomonas spp. The proteins naturally comprise 17 to 18 repeats of 34 amino acids. The binding specificity is determined by the amino acids at positions 12 and 13 within each repeat. Combined with an endonuclease, TALEs (referred to as TALENs) can be used to specifically target almost any known genomic sequence. The main difference between ZFNs and TALENs is the recognition of the DNA sequence. While ZFNs recognise nucleotide triplets, TALENs recognise single nucleotides, rendering TALENs, in theory, adjustable to any given sequence in a genome while ZFNs need defined prerequisites to be specific. TALENs have already been used to alter the genomes of rats, zebrafish, human iPSCs, and pigs (personal communication). Molecular scissors open a wide range of new applications for modifying the genome of different species or cells with which it has remained very difficult to work. Breeding for agricultural purposes and biomedicine, including the development of large animal models for human diseases and xenotransplantation, will greatly benefit from these new tools. With the advent of ZFN- and TALEN-mediated gene knockouts, mammalian transgenesis has taken a major leap forward as a straightforward technology for gene knockout and knock-in.

Parallel assembly for multiple site-directed mutagenesis of plasmids

A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants—larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction–ligation reaction.

High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Identification and characterization of protein phosphorylation sites often requires mass spectrometric analysis, which is not trivial or accessible to many laboratories. Here, a targeted strategy to mutagenize putative phosphorylation sites within mitogen-activated protein kinase (MAPK) substrates is described. This employs a combination of standard type II with type IIs restriction enzymes to rapidly create individual or multiple phosphorylation site mutant versions of kinase substrates with high efficiency, thereby reducing the cost for screening mutated clones.

Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

BackgroundThe TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.ResultsBarker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).ConclusionsThe DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.

PUC-IVT, a modified pUC19 based in vitro transcription vector

The popular cloning vector, pUC19, was modified into an in vitro transcription system, pUC-IVT, by incorporation of T7 promoter sequence and appropriate restriction sites for cloning of gene of interest and generation of transcript. Strategy was also devised to eliminate incorporation of undesirable sequences at both 5′ and 3′ ends of the transcript for its use in many sensitive downstream applications. All the major restriction sites, except SphI and PstI present in the MCS of pUC19, were retained in pUC-IVT for facile cloning. Any DNA fragment having 5′ blunt end, and cohesive 3′ end, generated by restriction enzymes present in MCS of pUC19, except SphI and PstI, can be directionally cloned into pUC-IVT backbone generated by restricting with StuI and the same restriction enzyme used for 3′-end generation of target DNA fragment. EcoRI digested modified gene coding for archaebacterial (Synechocystis sp.) precursor tRNA-glutamine (ptRNAGln), harbouring recognition site for a type IIS restriction enzyme, FokI, was ligated onto the StuI and EcoRI digested pUC-IVT to generate pUC-IVT::ptRNAGln. In vitro run-off transcription was performed using FokI digested linearized pUC-IVT::ptRNAGln plasmid DNA as template to get ptRNAGln transcript. Reconstituted Escherichia coli ribonuclease P (RNase P) efficiently cleaved 5′ leader sequence of ptRNAGln to generate mature tRNAGln.

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.

Simple cloning strategy using GFPuv gene as positive/negative indicator

Because construction of expression vectors is the first requisite in the functional analysis of genes, development of simple cloning systems is a major requirement during the postgenomic era. In the current study, we developed cloning vectors for gain- or loss-of-function studies by using the GFPuv gene as a positive/negative indicator of cloning. These vectors allow us to easily detect correct clones and obtain expression vectors from a simple procedure by means of the combined use of the GFPuv gene and a type IIS restriction enzyme.

Assembly of custom TALE-type DNA binding domains by modular cloning

Transcription activator-like effector (TALE) DNA binding proteins show tremendous potential as molecular tools for targeted binding to any desired DNA sequence. Their DNA binding domain consists of tandem arranged repeats, and due to this repetitive structure it is challenging to generate designer TALEs (dTALEs) with user-defined specificity. We present a cloning approach that facilitates the assembly of multiple repeat-encoding DNA fragments that translate into dTALEs with pre-defined DNA binding specificity. This method makes use of type IIS restriction enzymes in two sequential cut-ligase reactions to build dTALE repeat arrays. We employed this modular approach for generation of a dTALE that differentiates between two highly similar DNA sequences that are both targeted by the Xanthomonas TALE, AvrBs3. These data show that this modular assembly system allows rapid generation of highly specific TALE-type DNA binding domains that target binding sites of predefined length and sequence. This approach enables the rapid and flexible production of dTALEs for gene regulation and genome editing in routine and high-throughput applications.

A Modular Cloning System for Standardized Assembly of Multigene Constructs

The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.