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Transformation-associated recombination cloning (TAR)

Transformation-associated recombination (TAR) cloning in yeast is a technique to selectively isolate a gene sequence without the construction of a genomic library. It involves the recombination between the two ‘hooks’ of the TAR vector and the flanking sequence of the targeted gene. Application of TAR cloning includes the analysis of the disease causing genome of Trypanosoma brucei and also to fill in the gaps of the human genome. Future work includes the isolation of full length genes for the construction of human artificial chromosome (HAC)

Introduction:

The ability to clone and manipulate large stretches of DNA is one of the cornerstones of post-genomics molecular biology. To clone these large DNA fragments, yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) are employed and they been successful in furthering our understanding of complex genomes (Burke, et al., 1987). The use of conventional YAC and BAC to isolate a gene or specific chromosomal region is both time and labor intensive, and does not have any specificity for the desired DNA region (McGonigalet, et al., 1995). A genomic library is required containing thousands of sub clones and requiring multiple cloning steps to reassemble a copy of the target gene.

A technique called transformation-associated recombination (TAR) was developed in the late 1990s (Larionov, et al., 1996) which allows for the selective isolation of large DNA fragments from a complex genome without the construction of a genomic library. Under optimal conditions, fragments up to 250kb can be isolated from samples within 2 weeks (Kouprina & Larionov, 2006) with accuracy comparable to PCR. Until the development of TAR cloning, PCR was the only method to selectively isolate chromosomal fragments from a complex genome. PCR has a major drawback: DNA fragments larger than ~20kb cannot be amplified. TAR cloning solves this problem.

PDF link- topic-52-tar.pdf

Principles of TAR cloning

Figure one shows the general methodology in using TAR vector in cloning. A TAR vector consists of two ‘hooks’ at either end, which targets the desired gene flanking sequence, a centreomere sequence and a yeast selectable marker. The hooks can be a small as 60bp (Noskov, 2001). Both the TAR vector and human DNA is transformed into yeast spheroplasts. Recombination occurs between the hooks and the homologous region, resulting in a circular YAC that is able to be replicated and select for in yeast. Normally the yield with your gene of interest is very low (1-5%) but this can be increased to 30% by introducing double strand breaks (SSB) near the targeted regions.

In general terms, TAR can be classified into two categories (Kouprina, et al., 1998). If the sequence of the 3’ or 5’ flanking region of the targeted gene is known, than the gene is isolated with 2 unique sequence to form the two ‘hooks’ of the TAR vector. However, if the sequence of either the 3’ or 5’ flanking region is unknown, than a modified version of called radial TAR cloning is needed. In radial cloning, one hook still targets the known flanking sequence; the other hook targets a repeated sequence from the chromosome of interest that occurs frequently and randomly. In humans it’s the Alu or B1 repeats.

Figure 1 | The isolation of genes from human chromosomes using TAR cloning. A| The TAR vector consist of two ‘hooks’, which targets the flanking sequence of the gene of interest, a yeast centromere sequence (CEN) and a yeast selectable marker (HIS3). B|Both the vector and human DNA is transformed into yeast spheroplasts. C| The recombination between the ‘hooks’ and the homologous sequence (promoter region, 3’UTR) results in a circular YAC that is replicated and selected for in yeast. To confirm for positive gene insert, the diagnostic sequence is used. Source: Kouprina N, Larionov V, 2006

Applications of TAR cloning

The isolation of large stretches of genomic DNA is important in the field of functional genomics. The ability of TAR to not only selectively isolates the gene of interest, but also to clone its control elements (promoters, introns) in its ‘native’ form in the body, which makes studying gene function much easier. Previously the only way to get full-size gene was from a genomic library formed by YAC, BAC (Burke, et al., 1987).

Another application of TAR is for the analysis of highly variable chromosome in disease causing microbes. An example is through the analysis of the African trypanosomes which causes African sleeping sickness. It evades the immune system by switching its surface glycoprotein (VSGs). This variation in VSGs is due to the many bloodstream form VSG expression sites (BESs). To understand why the diversity of BES is important, a full sequence of BES is required for each particular strain. However, since most BES is located at telomere, cloning with conventional techniques has been difficult. However, with a modified TAR, it was possible to isolate an almost complete set of BES-containing telomeres from a particular strain (Marion, et al., 2004).

The completion of the human genome project in 2003 was major milestone in biology (Collins, et al., 2003). However, there are still gaps remaining that might take 5, 10 or even 20 years before each gap is filled (Eichler, et al., 2004). By using TAR cloning, problematic gaps sequences (ie sequences near the centreomere) can be cloned without the need for the construction of an entire genomic library. It is hoped that by completing the ‘complete’ genome, more information about diseases, evolution can be obtained.

Future of TAR cloning

For gene therapy to succeed, a delivery vehicle is needed to carry full length gene into cells. Current technology involves the use of viruses such as adeno viruses which illicit an immune response. One of the promising vectors is the Human artificial chromosome (HAC). Its advantage is that there are no viral proteins to trigger an immune response, and also because they are stable in low copy numbers in eh nucleus. The construction of these HACs can be made with the genomic sequences isolated from TAR. There are already several studies which show the efficiency of HAC as a delivery vehicle (Kakeda, 2005).

Conclusion

The development of TAR is an important tool in the armory of researchers. Not only does it shorten the time frame for the isolation of a gene, but it also allows for the selective cloning of products that is toxic to host systems such as in E.coli.

Works Cited

Burke, D. T., Carle, G. F., & Olson, M. V. (1987). Cloning of large segments of DNA into yeaqst by means of artifical chromosome vectors. Science , 806-812.

Burke, D. T., Carle, G. F., & Olson, M. V. (1987). Cloning of large segments of DNA into yeast by means of artifical chromosome vectors. Science , 806-812.

Collins, F. S., Green, E. D., Guttmacher, A. E., & Guyer, M. S. (2003). A version for the future of genomics research. Nature , 835-847.

Eichler, E. E., Clark, R. A., & She, X. (2004). An assessment of the sequence gaps: Unfinished bussiness in a finished human genome. Nature reviews , 345-354.

Kakeda, M. (2005). Human artifical chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human fibroblasts. Gene Ther , 852-856.

Kouprina, N., & Larionov, V. (2006). TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nature review: Genetics , 805-812.

Kouprina, N., Annab, L., Graves, J., Afshari, C., Barret, J. C., Resnick, M. A., et al. (1998). Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence. Proc Natl Acad Sci USA , 4469-4474.

Larionov, V., Kouprina, N., Graves, J., Chen, X., Korenberg, J. R., & Resnick, M. A. (1996). Specific cloning of human DNA as yeast artificial chromosome by transformation-associated recombination. Proc. Natl. Acad. Sci. USA , 491-496.

Marion, B., Niall, A., Elaine, B., Bill, W., Edward, L., & Gloria, R. (2004). Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast. Genome Research , 2319-2329.

McGonigal, T., Bodell, P., Schopp, C., & Sarthy, A. V. (1995). Construction of a human DNA library in a circular centromere based yeast plasmid. Gene , 267-271.

Noskov, V. (2001). Defining the minimal length of sequence fir selective gene isolation by TAR cloning. Nucleic Acids Res , e62.

edited 1st sept

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