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New yeast-bacteria shuttle vector

a shuttle vector and yeastbacteria technology, applied in the field of new yeastbacteria shuttle vectors, can solve the problems of cumbersome isolating sufficient quantities of yac dna from agarose gel for microinjection or electroporation, and affecting the efficiency of agarose gel agarose synthesis, and achieves the effect of high versatility of the cloning system

Inactive Publication Date: 2005-01-06
BRADSHAW M SUZANNE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] A new yeast-bacteria shuttle vector has been identified that allows selective cloning and / or manipulation of large regions of DNA. The vector of the present invention provides a unique combination of features including site-specific targeting, yeast to bacteria (and bacteria to yeast) shuttling capability, interchangeable recombinogenic ends, large insert capacity, and near universal compatibility with large insert cloning systems in bacteria and yeast. When combined with a method for direct cloning of co-transformed genomic DNA, the vector of the present invention provides a highly versatile cloning system.
[0011] The circular product of cloning in the present vector can be transferred to bacteria for simplified preparation of large quantities of plasmid DNA. The shuttle capability of the vector makes mutagenesis techniques by yeast genetics accessible for the production of large reporter constructs. The yeast bacterial shuttle vector of the present invention comprises a yeast replication origin, a yeast selection marker gene, a bacteria replication origin, a bacteria selection marker gene and at least one unique cloning site.

Problems solved by technology

However, these vectors are limited by their small insert capacity.
However, isolating sufficient quantities of YAC DNA from agarose gels for microinjection or electroporation remains cumbersome.
Purification remains a problem when the YAC comigrates with an endogenous chromosome.
These vectors are limited to bacteria and cannot be shuttled to yeast for modification by homologous recombination.

Method used

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Examples

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example 1

Construction of Shuttle Vector

[0040] The vector, pCLASPER. (FIG. 1), was constructed as follows. The PmlI site in pRS415 (Stratagene) was cleaved to insert a linker with a SacI site flanked on one side with a 10 bp reduplication of centromeric DNA from chromosome VI (CEN6) and with a random sequence on the other side to ascertain the orientation by PCR after cloning. The resultant plasmid was cleaved at the BamHI site in the polylinker of pRS415 and at a single DraIII site between the polylinker and the beta-isopropyl malate dehydrogenase gene (LEU2) to insert a 100 bp synthetic DNA fragment containing part of a newly designed polylinker. The new plasmid was digested with Sacl and BamHI to isolate the CEN6 / ARS4 origin of replication, the LEU2 gene, and one half of the new polylinker. Plasmid pBCSK (Stratagene) was used as a substrate to generate by PCR a 1103 bp fragment encompassing, the chloramphenicol acetyl transferase (Cmr) gene from Tn9. The PCR primer for the 5′ end of the g...

example 2

Construction of Targeting Vectors

[0041] The YACs to be targeted were obtained from the Princeton mouse genomic YAC library (6). Isolation and characterization of the Hoxb and Hoxc cluster YACs have been previously described (7, 8). The targeting vector designed to clone the Hoxc-8 gene, pClC9C6, was made as follows. PCR primers for the Hoxc-6 gene (9) were designed to the 5′ untranslated region of the gene. The 5′ primer was: 5′-TAGATCTGTTTGTCTCCCACATGCC-3′ and contained a Nrul linker. The 3′ primer was: 5′-AGGTGGCAGGATAAGGAAGGGTTAG-3′ and contained a HindIII linker. The product was 706 bp. The PCR primers for the Hoxc-9 gene (10) were designed to amplify a 624 bp fragment of the 3′ untranslated region. The 5′ primer was: 5′-CGACAAGGAACAAATCCTAAGCCC-3′ and contained a BamHI linker. The 3′ primer was: 5′-TGCATTTGCAGCCTGATCCAGCCA-3′ and contained a NruI linker. All polymerase chain reactions (PCR) was carried out in a Hybard Omnigene machine. Reactions contained 1× Boehringer Mannhei...

example 3

Preparation of DNA for Yeast Transformations

[0045] Since pCLASPER is maintained at single copy, the following procedures facilitate the preparation of large quantities of plasmid DNA. E. coli strain DH1OB cells containing the targeting vectors were grown overnight in 1-2 liters TB media containing 20 μg / ml chloramphenicol. Each one liter culture was divided into 3 centrifuge bottles to pellet. The Plasmid Maxi Protocol for Qiagen-tip 500 (Qiagen) was followed for each pellet with the following changes. After the addition of 10 ml each of buffers P1, P2 and P3 to each pellet, the suspension was centrifuged at 4° C. for 30 minutes at 15,000×g. The supernatant was removed, then recentrifuged under the same conditions. The supernatants were pooled, passed through a 0.45 micron filter and loaded on a single Qiagen-tip 500 following the suppliers directions. Generally, at least 20 μg vector DNA per liter of cells was obtained as determined by comparing both optical density and visual est...

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Abstract

The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described, that can be used to clone large regions of DNA by homologous recombination. The important feature of present invention is the presence of the a bacterial replication origin, which allows large DNA insert capacity. The utility of this vector lies in its ability to isolate, manipulate and maintain large fragments in bacteria and yeast, allowing for mutagenesis by yeast genetics and simplified preparation of plasmid DNA in bacteria.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the field of genomics and the ability to manipulate, isolate and replicate large fragments of DNA. BACKGROUND OF THE INVENTION [0002] For the functional analysis of many genes investigators need to isolate and manipulate large DNA fragments. The advent of genomics and the study of genomic regions of DNA has generated a need for vectors capable of carrying large DNA regions. [0003] In general, two types of yeast vector systems are presently available. The first type of vector is one capable of transferring small insert DNA between yeast and bacteria (33, 34). A second type of vector is a fragmenting vector which creates interstitial or terminal deletions in YACs (35, 36, 37). The small insert shuttle vectors are able to recombine with and recover homologous sequences. They are centromere-based and replicate stably and autonomously in yeast, but also contain a high-copy origin of replication for maintenance as bacterial pl...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N1/21C12N15/10C12N15/70C12N15/81C12Q1/68
CPCC12N15/1034C12N15/70C12Q1/6827C12Q1/68C12N15/81
Inventor BRADSHAW, M. SUZANNEBOLLEKENS, JACQUES A.RUDDLE, FRANK H.
Owner BRADSHAW M SUZANNE
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