Vector Arms Research Articles

Yeast artificial chromosome vectors for efficient clone manipulation and mapping

The yeast artificial chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length. To enhance the usefulness of this technology, yeast artificial chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping. The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends. Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli. In addition, YACs generated using this vector system contain a yeast gene ( SUP11) that allows visual monitoring of YAC stability and copy number.

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.

Second-generation approach to the construction of yeast artificial-chromosome libraries

We describe an improved method for construction of yeast artificial-chromosome (YAC) libraries that contain large inserts of foreign DNA. The procedure consists of seven steps: (i) preparation of human DNA in agarose beads; (ii) partial digestion of the DNA with EcoRI; (iii) electrophoretic elimination of the smaller partial-digest fragments; (iv) ligation of the EcoRI fragments with vector arms in molten agarose; (v) hydrolysis of agarose with agarase; (vi) fractionation of the recombinant molecules by sucrose-gradient centrifugation; and (vii) transformation of yeast. More than 7000 colonies were obtained starting with 15 μg of human DNA, which was fractionated on a single sucrose gradient. The average size of these YACs was approximately 380 kb. It is estimated that the total length of human DNA present in the clones corresponds to 80% of the length of the human haploid genome. The results of screening the clones for a number of single-copy genes indicate that the clones reflect a nearly random sampling of the human genome. The efficiency of the cloning is sufficient to support the construction of multihit libraries for the human genome or for the genomes of other higher organisms.

An efficient directional cloning system to construct cDNA libraries containing full-length inserts at high frequency

We have developed a high efficiency cDNA cloning system which can direct the orientation of inserts in λ-plasmid composite vectors with large cloning capacities. Cleavage of the vector DNA by SfiI creates two different nonsymmetrical 3′ extensions at the ends of the vector arms. Using a linker-primer and an adaptor, cDNA is prepared so it has two different sticky ends which can be ligated to those of the vector arms. When the cDNA fragments and the vector arms are mixed, both the molecules can assemble without self-circularization due to base-pairing specificity. Ligation of the cDNA-vector mixture produces the concatemers from which phage clones carrying a single cDNA insert in the desired orientation can be formed very efficiently by in vitro packaging. This system provides: (1) high cloning efficiency [10 7−10 8 clones/μg poly(A) +RNA], (2) low background (more than 90% of the clones contain inserts), (3) directional insertion of cDNA fragments into the vectors, (4) presence of a single insert in each clone, (5) accommodation of long inserts (up to 10 kb), (6) a mechanism for rescue of the plasmid part from the λ genome, and (7) a straightforward protocol for library preparation. Screenings of cDNA libraries constructed by this method demonstrated that cDNAs of up to 6.4 kb, containing complete coding sequences, could be isolated at high efficiency. Thus, this cloning system should be useful for the isolation of cDNAs of relatively long transcripts, present even at low abundance, in cells.

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

We constructed a series of cosmid vectors that carry the two cohesive end sites ( cos) of λ 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, sizeselected 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 × 10 6−1.3 × 10 7 colony forming units (cfu)/μg of insert DNA and 1.7 × 10 5−1.0 × 10 6 cfu/μg 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.

Size Fractionation Using Sucrose Gradients

DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.

Charons 36 to 40: multi enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers.

New phage lambda based cloning vectors, Charons 36-40, have been constructed which allow cloning of large (up to 24 kb) DNA fragments with up to sixteen cloning enzymes. Several of these could not be used previously with lambda vectors. Clones produced with these vectors can be propagated under recombination deficient conditions. A novel polystuffer method has been developed that permits vector arms to be purified by simple precipitation and which allows reliable identification of clones that have reincorporated any part of the stuffer. Three of the vectors are available with amber mutations in essential genes.

Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.

Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.

Cloning of genomic sequences from the human Y chromosome after purification by dual beam flow sorting.

Human Y chromosomes were purified by dual beam flow sorting from a human X Chinese hamster cell line retaining the Y as the only free human chromosome. DNA was extracted from the Y fraction and cloned into lambda gtWES . lambda B vector arms. More than 100 recombinant clones carrying human inserts have been characterised by Benton-Davis plaque screening and Southern blotting or in situ hybridisation. Several repetitive sequences were found to be predominantly located on the Y, whereas the majority also cross-hybridised with autosomal DNA. One repetitive clone gave a specific hybridisation signal with the X and the Y chromosome but not with autosomes. Preliminary evidence indicates that many clones contain single copy as well as repetitive sequences. However, no Y-specific single copy sequence has yet been identified.

1] New bacteriophage lambda vectors with positive selection for cloned insects

Publisher Summary This chapter discusses new bacteriophage lambda vectors with positive selection for cloned inserts. Molecular cloning methods eliminated the necessity for physical fractionation of DNA and permitted, for the first time, the isolation of eukaryotic structural genes. Most bacteriophage vectors are substitution vectors that require internal filler fragments to be physically separated from the vector arms before insertions of foreign DNA. Genomic DNA is partially digested with restriction endonucleases to produce a population of DNA fragments from which molecules 15–20 kb long are purified by agarose gel electrophoresis. Other derivatives of 1059 that introduce defined amber mutations or alter the restriction enzyme sites on the vector have also been constructed. Successful and efficient cloning requires highly purified restriction enzymes and active DNA ligase.

Some Navigational Techniques for Use with Marine Radar

The parallel cursor and disc. In the Royal Navy manœuvring problems are normally worked out on the Battenburg Course Indicator which gives a mechanical solution of the speed/distance triangle (Fig. 1). A similar device for direct use on the PPI face can be made by engraving parallel lines on the rotatable PPI mask and attaching two perspex vector arms to the centre. Although developed primarily for manœuvring purposes this idea can be put to several purely navigational uses and from it have sprung two valuable adjuncts to the PPI: the parallel cursor and the fixed plotting surface (or disc).