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De novo assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats.

Lin L, Koo DH, Zhang W, St Peter J, Jiang J - Plant Methods (2011)

Bottom Line: The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli.Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA. jjiang1@wisc.edu.

ABSTRACT

Background: Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.

Results: We report a novel approach to assemble AC constructs that are capped with long telomeric DNA. We designed a plasmid vector that can be combined with a bacterial artificial chromosome (BAC) clone containing centromeric DNA sequences from a target plant species. The recombined clone can be used as the centromeric DNA backbone of the AC constructs. We also developed two plasmid vectors containing short arrays of plant telomeric DNA. These vectors can be used to generate expanded arrays of telomeric DNA up to several kilobases. The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.

Conclusions: We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli. Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

No MeSH data available.


Related in: MedlinePlus

Generation of back-to-back telomeric DNA fragments. (A) Size-fractionated telomeric DNA fragments ranging in size from 4 to 10-kb derived from pLL-TBS and pLL-TSB, respectively. (B) An example of a 6-kb back-to-back telomeric DNA combined from two 3-kb telomeric DNA samples derived from pLL-TBS and pLL-TSB, respectively. The 3-kb band is the non-recombined telomeric DNA.
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Figure 3: Generation of back-to-back telomeric DNA fragments. (A) Size-fractionated telomeric DNA fragments ranging in size from 4 to 10-kb derived from pLL-TBS and pLL-TSB, respectively. (B) An example of a 6-kb back-to-back telomeric DNA combined from two 3-kb telomeric DNA samples derived from pLL-TBS and pLL-TSB, respectively. The 3-kb band is the non-recombined telomeric DNA.

Mentions: Digesting the pLL-TBS and pLL-TSB vectors with BsgI released the short telomeric DNA inserts, including the attB1 site (Figure 2). The released DNA fragments were used as templates to generate long telomeric DNA fragments by unidirectional replication. This amplification step was accomplished by using Vent DNA polymerase that can catalyze short repeat expansion [16]. DNA fragments in the range of 2-10 kb were readily amplified using this approach (data not shown). The amplified telomeric DNA fragments were size-fractionated via gel excision to generate telomeric DNA varying in lengths (Figure 3A). The 5'-(TTTAGGG)n-3' and 3'-(TTTAGGG)n-5' DNA fragments of 2 to 5-kb in size were digested with I-SceI and ligated to form back-to-back telomeric DNA (Figure 2, Figure 3B). Because the homing endonuclease I-SceI recognizes asymmetric sites and the I-SceI sites on the pLL-TBS and pLL-TSB seed vectors are arranged in an opposite orientation, a telomeric DNA fragment derived from pLL-TBS will only ligate with a fragment derived from pLL-TSB. Thus, the resultant back-to-back telomeric DNA will include two 2 to 5-kb synthetic telomeric DNA fragments in opposite orientation, one I-SceI site, and one attB1 site (Figure 2).


De novo assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats.

Lin L, Koo DH, Zhang W, St Peter J, Jiang J - Plant Methods (2011)

Generation of back-to-back telomeric DNA fragments. (A) Size-fractionated telomeric DNA fragments ranging in size from 4 to 10-kb derived from pLL-TBS and pLL-TSB, respectively. (B) An example of a 6-kb back-to-back telomeric DNA combined from two 3-kb telomeric DNA samples derived from pLL-TBS and pLL-TSB, respectively. The 3-kb band is the non-recombined telomeric DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101654&req=5

Figure 3: Generation of back-to-back telomeric DNA fragments. (A) Size-fractionated telomeric DNA fragments ranging in size from 4 to 10-kb derived from pLL-TBS and pLL-TSB, respectively. (B) An example of a 6-kb back-to-back telomeric DNA combined from two 3-kb telomeric DNA samples derived from pLL-TBS and pLL-TSB, respectively. The 3-kb band is the non-recombined telomeric DNA.
Mentions: Digesting the pLL-TBS and pLL-TSB vectors with BsgI released the short telomeric DNA inserts, including the attB1 site (Figure 2). The released DNA fragments were used as templates to generate long telomeric DNA fragments by unidirectional replication. This amplification step was accomplished by using Vent DNA polymerase that can catalyze short repeat expansion [16]. DNA fragments in the range of 2-10 kb were readily amplified using this approach (data not shown). The amplified telomeric DNA fragments were size-fractionated via gel excision to generate telomeric DNA varying in lengths (Figure 3A). The 5'-(TTTAGGG)n-3' and 3'-(TTTAGGG)n-5' DNA fragments of 2 to 5-kb in size were digested with I-SceI and ligated to form back-to-back telomeric DNA (Figure 2, Figure 3B). Because the homing endonuclease I-SceI recognizes asymmetric sites and the I-SceI sites on the pLL-TBS and pLL-TSB seed vectors are arranged in an opposite orientation, a telomeric DNA fragment derived from pLL-TBS will only ligate with a fragment derived from pLL-TSB. Thus, the resultant back-to-back telomeric DNA will include two 2 to 5-kb synthetic telomeric DNA fragments in opposite orientation, one I-SceI site, and one attB1 site (Figure 2).

Bottom Line: The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli.Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA. jjiang1@wisc.edu.

ABSTRACT

Background: Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.

Results: We report a novel approach to assemble AC constructs that are capped with long telomeric DNA. We designed a plasmid vector that can be combined with a bacterial artificial chromosome (BAC) clone containing centromeric DNA sequences from a target plant species. The recombined clone can be used as the centromeric DNA backbone of the AC constructs. We also developed two plasmid vectors containing short arrays of plant telomeric DNA. These vectors can be used to generate expanded arrays of telomeric DNA up to several kilobases. The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.

Conclusions: We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli. Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

No MeSH data available.


Related in: MedlinePlus