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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

Visualization of linear AC constructs by fiber-FISH using pLL-EHC (green) and telomeric DNA (red), as probes. The two arrows of the same color in the four different images (A, B, C, and D) point to the two telomeric signals on the same AC constructs. Arrowheads point to some of the non-recombined circular pLL-EHC molecules. Note: the variability in size of the observed linear and circular DNA molecules is caused by non-uniform extension of the DNA molecules. Bars = 10 μm.
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Figure 6: Visualization of linear AC constructs by fiber-FISH using pLL-EHC (green) and telomeric DNA (red), as probes. The two arrows of the same color in the four different images (A, B, C, and D) point to the two telomeric signals on the same AC constructs. Arrowheads point to some of the non-recombined circular pLL-EHC molecules. Note: the variability in size of the observed linear and circular DNA molecules is caused by non-uniform extension of the DNA molecules. Bars = 10 μm.

Mentions: We used a DNA fiber-fluorescence in situ hybridization (fiber-FISH) technique to visualize the AC constructs resulting from the ligations between pLL-EHC and 4 to 8-kb back-to-back telomeric DNA fragments. The recombined DNA was directly spread on poly-lysine coated glass slides and hybridized with telomeric DNA probe (red) and pLL-EHC (green) probes. Linear DNA molecules hybridized with both probes were consistently detected using DNA samples from different ligation experiments (Figure 6). Non-recombined and circular pLL-EHC molecules were also observed. Some linear molecules showed no telomeric DNA signals or a signal at only one of the two ends. However, this is likely due to the resolution limitations of the fiber-FISH technique in which DNA sequences as short as few kilobases are often not detected as consistently as longer DNA fragments.


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)

Visualization of linear AC constructs by fiber-FISH using pLL-EHC (green) and telomeric DNA (red), as probes. The two arrows of the same color in the four different images (A, B, C, and D) point to the two telomeric signals on the same AC constructs. Arrowheads point to some of the non-recombined circular pLL-EHC molecules. Note: the variability in size of the observed linear and circular DNA molecules is caused by non-uniform extension of the DNA molecules. Bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Visualization of linear AC constructs by fiber-FISH using pLL-EHC (green) and telomeric DNA (red), as probes. The two arrows of the same color in the four different images (A, B, C, and D) point to the two telomeric signals on the same AC constructs. Arrowheads point to some of the non-recombined circular pLL-EHC molecules. Note: the variability in size of the observed linear and circular DNA molecules is caused by non-uniform extension of the DNA molecules. Bars = 10 μm.
Mentions: We used a DNA fiber-fluorescence in situ hybridization (fiber-FISH) technique to visualize the AC constructs resulting from the ligations between pLL-EHC and 4 to 8-kb back-to-back telomeric DNA fragments. The recombined DNA was directly spread on poly-lysine coated glass slides and hybridized with telomeric DNA probe (red) and pLL-EHC (green) probes. Linear DNA molecules hybridized with both probes were consistently detected using DNA samples from different ligation experiments (Figure 6). Non-recombined and circular pLL-EHC molecules were also observed. Some linear molecules showed no telomeric DNA signals or a signal at only one of the two ends. However, this is likely due to the resolution limitations of the fiber-FISH technique in which DNA sequences as short as few kilobases are often not detected as consistently as longer DNA fragments.

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