Efficient isothermal expansion of human telomeric and minisatellite repeats by Thermococcus litoralis DNA polymerase

Hartig, Jörg S.; Kool, Eric T.

doi:10.1093/nar/gki803

Cited by 6 publications

(8 citation statements)

References 38 publications

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“…A thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase) was used to generate long telomeric repeats from a short synthetic template/primer following a previously published protocol [ 16 ]. We cloned a 340-bp telomeric repeat fragment into the pGEM-T Easy vector.…”

Section: Resultsmentioning

confidence: 99%

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

Koo

Zhang

et al. 2011

Plant Methods

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BackgroundArtificial 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.ResultsWe 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.ConclusionsWe 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.

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Section: Resultsmentioning

confidence: 99%

“…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).…”

Section: Resultsmentioning

confidence: 99%

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

Koo

Zhang

et al. 2011

Plant Methods

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“…[34] Long DNA strandsc ontaining high copy numbers( up to 10 kbp) of short repeats equences (2-3 nucleotides)a re readily synthesised from 10-20 mer duplex oligonucleotides using isothermals lippage methods. [17,35] Although isothermal slippage has been shown to occur for 6-10 bp repeats such as telomeric sequences, [36] we have found that intermediate sized repeat sequences (4-40 nucleotides)a re most efficiently extended by thermalc ycling methods. [37] Moreover,i ti sd ifficult to produce high copy number repeat-sequence DNA containing modified nucleotides by isothermal extension, even for short repeat sequences and low density modifications.…”

Section: Introductionmentioning

confidence: 89%

Self‐Priming Enzymatic Fabrication of Multiply Modified DNA

Whitfield

Little

Khan

et al. 2018

Chemistry A European J

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The self-priming synthesis of multiply modified DNA by the extension of repeating unit duplex "oligoseeds" provides a source of versatile DNA. Sterically-demanding nucleotides 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP as well as 5-(octadiynyl)-dCTP were incorporated into two extending oligoseeds; [GATC] /[GATC] and [A G] /[CT ] . The products contained modifications on one or both strands of DNA, demonstrating their recognition by the polymerase as both template (reading) and substrate (writing). Nucleobase modifications that lie in the major groove were reliably read and written by the polymerase during the extension reaction, even when bulky or in contiguous sequences. Repeat sequence DNA over 500 bp long, bearing four different modified units was produced by this method. The number, position and type of modification, as well as the overall length of the DNA can be controlled to yield designer DNA that offers sequence-determined sites for further chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.

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“…This challenge can be partly surmounted by conducting the synthesis at elevated temperatures (70–80 °C) using thermostable Vent DNA polymerase. At high temperatures, the product of the polymerase reaction can be relatively long, although not exceeding 1 kbp for palindromic, , telomeric, and minisatellite repeats.…”

mentioning

confidence: 99%

Multiply Modified Repeating DNA Templates for Production of Novel DNA-Based Nanomaterial

et al. 2019

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Here, we report synthesis of long (thousands of base pairs), uniform double-stranded (ds) DNA comprising short (6−15 base pairs) tandem repeats. The synthesis method is based on self-assembly of short (6− 15 bases) half-complementary 5′-end phosphorylated single-stranded oligonucleotides into long ds polymer molecules and covalent association of the oligonucleotide fragments in the polymer by DNA ligase to yield complete non-nicked ds DNA. The method is very flexible in regard to the sequence of the oligonucleotides and their length. Human telomeric DNA comprising thousands of base pairs as well as methylated, mismatched, and fluorescent dye-modified uniform dsDNA molecules can be synthesized. We have demonstrated by high resolution frequency-modulation atomic force microscopy that the structure of DNA containing mismatches is strongly different from that of the non-mismatched one. The DNA molecules comprising groups capable of anchoring metal particles and other redox active elements along the whole length of the nucleic acid polymer should find use as wires or transistors in future nanoelectronic applications.

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Efficient isothermal expansion of human telomeric and minisatellite repeats by Thermococcus litoralis DNA polymerase

Cited by 6 publications

References 38 publications

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

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

Self‐Priming Enzymatic Fabrication of Multiply Modified DNA

Multiply Modified Repeating DNA Templates for Production of Novel DNA-Based Nanomaterial

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