Rapid one-step recombinational cloning

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning.     

Mark–recapture cloning: a straightforward and cost‐effective cloning method for population genetics of single‐copy nuclear DNA sequences in diploids

We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′‐tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual‐based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high‐throughput method, providing the number of sequences exceeds the number of individuals initially amplified.     

Enzyme Free Cloning for high throughput gene cloning and expression

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.     

Rapid and efficient cosmid cloning

We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

IRDL Cloning: A One-Tube,Zero-Background,Easy-to-Use,Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation

T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.     

Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.     

Data cloning: easy maximum likelihood estimation for complex ecological models using Bayesian Markov chain Monte Carlo methods

We introduce a new statistical computing method, called data cloning, to calculate maximum likelihood estimates and their standard errors for complex ecological models. Although the method uses the Bayesian framework and exploits the computational simplicity of the Markov chain Monte Carlo (MCMC) algorithms, it provides valid frequentist inferences such as the maximum likelihood estimates and their standard errors. The inferences are completely invariant to the choice of the prior distributions and therefore avoid the inherent subjectivity of the Bayesian approach. The data cloning method is easily implemented using standard MCMC software. Data cloning is particularly useful for analysing ecological situations in which hierarchical statistical models, such as state-space models and mixed effects models, are appropriate. We illustrate the method by fitting two nonlinear population dynamics models to data in the presence of process and observation noise.     

Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.

We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.     

外切核酸酶Ⅲ介导的DNA分子克隆

DNA克隆技术,作为最基本的现代分子生物学实验技术之一, 已经成为生物医学研究领域的重要研究手段。传统的分子克隆方法需要经过限制性内切酶酶切和DNA连接酶连接的步骤,是否存在合适的酶切位点和DNA连接酶的效率成为影响克隆的重要限制因素。本文描述了一种由外切核酸酶Ⅲ介导的,以3′-5′外切核酸酶活性和细菌细胞内DNA修复机制为理论基础的DNA分子克隆方法,称为不依赖连接酶的分子克隆（ligation-independent cloning, LIC）|证明了该方法的高效性和可靠性,并进一步对酶的用量、反应温度、反应时间、片段载体比例和量等多个参数进行了优化,建立了一种快速、简便和高效的DNA克隆方法。     

An advanced blue-white screening method for construction of shRNA expression vectors

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.     

A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses.

We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.     

Restriction cutting independent method for cloning genomic DNA segments outside the boundaries of known sequences

We present a simple method for cloning genomic DNA segments outside the boundaries of known sequences, which is not dependent on restriction cutting or mapping. In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments are separated by cloning and characterized by sequencing. The method was used to clone both the upstream (5') and the downstream (3') genomic regions of an intron-interrupted tRNA(Leu)(UAA) gene from three cyanobacteria belonging to the genus Microcystis.     

Direct cloning by covalent attachment of probe DNA to target DNA.

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Identification of procaryotic repetitive DNA suitable for use as fingerprinting probes.

We have developed a method which enables the cloning and identification of procaryotic repetitive DNA suitable for use as DNA fingerprinting probes. The method involves shotgun cloning of restricted genomic DNA with subsequent selection of clones containing repetitive DNA by reverse-probed genomic hybridizations, in which the plasmid DNA clones are probed with labelled genomic DNA. Confirmation that the clones contained repeated sequences was by Southern hybridization, gene copy equivalence, and DNA sequencing. The sequences were used for highly specific and sensitive detection of bacteria and as target sequences for the mediation of chromosomal integration of reporter gene constructs.     

A one pot, one step, precision cloning method with high throughput capability

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.