USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.     

Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA. Recombinants are introduced into cells conventionally by transformation or electroporation. In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.     

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.     

Self-formed adaptor PCR: a simple and efficient method for chromosome walking

We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb. SEFA PCR is simple and efficient and should have broad applications in the isolation of unknown sequences in complex genomes.     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.     

An efficient method for purification of PCR products for sequencing

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees.     

A simple,efficient, standard-dilution method for cloning hybridomas

Summary A simple standard-dilution method which obviates routine use of viable cell counts, feeder layers and time-consuming scale-up procedures is described. This method can be used to clone monoclonal antibody-producing hybridomas 9–14 days post-fusion. Each cloning cycle takes 5 min. Approximately 60% of the positive, monoclonal antibody-producing hybridomas were successfully cloned and established.     

A simple,efficient method for purification of degraded PCR primers

Summary A simple, efficient method was used to purify PCR primers which had degraded during storage at-20°C/-9°C. The primers were eiectrophoresed on 3% (w/v) agarose gel, the main band was electroeluted via a trough cut in the gel. The primers were recovered by isobutanol extraction followed by ethanol precipitation. The yield was 20–40% and the A₂₆₀/A₂₈₀ ratio was greater than 1.8. The purification resulted in good amplification.     

A simple and eficient method for high fidelity PCR cloning using antibody-neutralizing technology

We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.     

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.     

A simple and fast method for cloning and analyzing polymerase chain reaction products

A method describing a fast and efficient way for cloning polymerase chain reaction (PCR) products is presented that involves end repair and purification of the PCR product, followed by kinasing and ligation to the vector with the use of a temperature gradient. Efficiency of ligation was estimated to be 50%–70%. Following transformation, cells are plated on MacConkey agar. Bacteria from selected colonies are used directly from the plates for screening without any subsequent purification. Using this protocol, PCR products can be efficiently cloned quickly and economically.     

一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

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.     

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.     

An efficient and reliable method for cloning PCR-amplification products: a survey of point mutations in integrin cDNA.

A highly efficient, non-labor-intensive method for cloning DNA fragments produced by PCR amplification was used to carry out a rapid survey of potential point mutations in integrin alpha 6 cDNA from 17 different cell-type sources. The method includes glass powder purification of the PCR reaction mixture, followed by simultaneous treatment with T4 polynucleotide kinase and DNA polymerase I, and another glass powder purification. Sequences from multiple subclones of each cell type were readily generated, aligned and checked for mismatches. Several commonly used alternative procedures were compared for cloning efficiency and size-fidelity of inserted DNA fragments.     

A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously

Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.