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.     

New cloning vectors and techniques for easy and rapid restriction mapping

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

Direct cloning of a long restriction fragment aided with a jumping clone.

Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.     

Direct Cloning and Sequencing of Bacterial Artificial Chromosome (BAC) Insert Ends Based on Double Digestion

Conventional digestion and ligation was developed into a novel and efficient approach for directly cloning and sequencing the two ends of bacterial artificial chromosome (BAC) clone inserts. Most BAC vectors have two Not I sites. This end isolation method is based on double digestion of the BAC clone DNA with Not I and any blunt-end restriction enzyme for which there is not a restriction site located within the small fragment (containing the cloning site) between the two Not I sites on the BAC vector. Digestion is followed by ligation of the double-digested mixture with a suitable plasmid vector. The pBeloBAC11 and pBlueScriptII SK vectors were used in the present study. The two ends of the BAC insert can be amplified and sequenced with three specific primers, i.e., amplification of the left end with the pBeloBAC11 LF1 and pBlueScriptII KS primers, and the right end with the pBeloBAC11 RR4 and KS primers. They may be directly recovered by transformation if the end fragments are used as probes. More significantly, this simple strategy generally can be applied to any BAC vector with any cloning site.     

A phage T4 in vitro packaging system for cloning long DNA molecules.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.     

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.     

Assisted large fragment insertion by Red/ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering

Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4-5 kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a approximately 55 kb fragment from a BAC clone containing the human Lhcgr gene into a 170 kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments.     

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.     

Rapid and reliable dideoxy sequencing of double-stranded DNA

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M 13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.     

Efficient simplified cosmid cloning: construction and characterization of cosmid vectors that carry the two cohesive end sites of lambda phages arrayed in tandem

We constructed a series of cosmid vectors that carry the two cohesive end sites (cos) of lambda phage, arrayed in tandem, which enabled us to clone fragments of genomic DNA of up to 50 kb without a vector background. An equimolar mixture of the left and right vector arms of equal length was prepared from the vector DNA, simply by treating the DNA sequentially with three enzymes, restriction enzyme PvuII, alkaline phosphatase, and restriction enzyme BamHI (or BglII), without purification by agarose gel electrophoresis. After phenol extraction and ethanol precipitation, the equimolar mixture of the vector arms, which carried a single cos oriented from left to right, was directly ligated with insert DNA without further manipulation. We established conditions for cosmid cloning, using two kinds of DNA fragment of 40-50 kb, prepared from mouse L cell genomic DNA, as insert DNAs, namely, three cloned BamHI fragments and Sau3AI fragments, size-selected on a sucrose density gradient. The most important parameters affecting the cloning efficiency were the quality of the insert DNA and the molar ratio of the insert and vector arms. We achieved cloning efficiencies of 3.6 X 10(6)-1.3 X 10(7) colony forming units (cfu)/micrograms of insert DNA and 1.7 X 10(5)-1.0 X 10(6) cfu/micrograms of insert DNA, using the cloned BamHI fragments and the Sau3AI fragments, respectively. We examined more than 5000 clones and found that they all contained insert DNA.     

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%.     

Optimized protocols and plasmids for in vivo cloning in yeast

Saccharomyces cerevisiae has proven a valuable system for the construction of plasmids via gap repair or in vivo cloning. The method allows cloning with superior accuracy and without the need to use restriction enzymes. However, despite its remarkable efficiency, the process may occasionally require the screening of large number of candidates. We have previously reported that by simply using shuttle plasmids that allow blue/white selection in Escherichia coli, it is possible to pre-select for positive clones. Here, we demonstrate that the same strategy can be used to assemble plasmids from several ectopic DNA fragments, which are all introduced in yeast cells by a simple transformation step. Further, to facilitate the subcloning of the fragment cloned into other targeting or expression vectors, the multi-cloning sites of three shuttle plasmids have been extended to include fifteen new restriction enzyme recognition sites.     

Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments

A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI. Plasmid pNIMB does not differ from the parent one phenotypically. Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments. Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel. Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used.     

Cloning and characterization of the termination site tI for the gene int transcript in phage lambda

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.     

Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.     

High efficiency vectors for cosmid microcloning and genomic analysis

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.