In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I

Kotlyar, Alexander B.

doi:10.1093/nar/gki178

Cited by 57 publications

(79 citation statements)

References 36 publications

(35 reference statements)

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“…Here, we present STS results on long homogeneous single novel poly(G)-poly(C) DNA molecules 28 deposited on gold 13 : such results enable us for the first time to resolve conductance peaks that are associated with the electronic levels of the nucleic acids. In parallel, insight into the nature of such levels is gained by ab initio DFT calculations for the very same base sequence, eventually enabled by the existence of clear experimental data.…”

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confidence: 99%

Electronic structure of single DNA molecules resolved by transverse scanning tunnelling spectroscopy

Shapir

Cohen

Calzolari³

et al. 2007

Nature Mater

147

176

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Attempts to resolve the energy-level structure of single DNA molecules by scanning tunnelling spectroscopy span over the past two decades, owing to the unique ability of this technique to probe the local density of states of objects deposited on a surface. Nevertheless, success was hindered by extreme technical difficulties in stable deposition and reproducibility. Here, by using scanning tunnelling spectroscopy at cryogenic temperature, we disclose the energy spectrum of poly(G)-poly(C) DNA molecules deposited on gold. The tunnelling current-voltage (I-V ) characteristics and their derivative (dI/dV -V ) curves at 78 K exhibit a clear gap and a peak structure around the gap. Limited fluctuations in the I-V curves are observed and statistically characterized. By means of ab initio density functional theory calculations, the character of the observed peaks is generally assigned to groups of orbitals originating from the different molecular components, namely the nucleobases, the backbone and the counterions.The electrical properties of single double-stranded DNA molecules have attracted great interest in the past two decades, leading to a series of experiments 1-3 to study the electron transfer and conduction through single DNA molecules and in various aggregation forms 4,5 . Nearly all of the single-molecule experiments addressed the conductivity along molecules that are attached to electrodes at the molecule ends. Besides fixing many chemical and physical properties, the intrinsic electronic structure of an object also determines its response to an external electric field. Hence, it is desirable to get this knowledge for DNA to understand its suitability to support electrical currents and the viable transport mechanisms. Scanning tunnelling spectroscopy (STS) is the technique of choice for measuring the electronic density of states (DOS) of a single molecule 6 , as was demonstrated through the years for various carbon nanostructures 7,8 , molecular objects 9 and inorganic nanoparticles 10 deposited on substrates.Following the invention of the scanning tunnelling microscope (STM) in 1982, there was a substantial number of attempts to measure single DNA molecules using STM. However, whereas several groups showed high-resolution images with quite detailed structure of the DNA molecules [11][12][13][14][15][16][17][18] , direct tunnelling spectroscopy across the helices was reported only a few times 18-21 and clear interpretation was inhibited by technical hurdles 22 . In all of these studies, DNA-salt aggregates or very short DNA oligomers with no clear orientation were measured. Moreover, the STS measurements of DNA were always done at room temperature, making it impossible to resolve the electronic levels within the thermal noise.Theoretical predictions for the DNA electronic structure, commonly based on molecular quantum mechanics calculations such as density functional theory 23,24 (DFT) or Hartree-Fock 25 , cannot be conclusive. In fact, the lack of clear experimental data for single DNA molecules hinders the...

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confidence: 99%

Electronic structure of single DNA molecules resolved by transverse scanning tunnelling spectroscopy

Shapir

Cohen

Calzolari³

et al. 2007

Nature Mater

147

176

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“…Compared with other enzymes used previously (12,14,16), Vent DNA polymerase is more efficient both in terms of product quantity (as judged by fraction of primer extension) and by length of the products. In addition, the products are characterized by a narrow length distribution.…”

Section: Discussionmentioning

confidence: 80%

“…For example, the expansion of telomeric sequences has been studied by Yoshida and co-workers (15), who demonstrated that a DNA polymerase α/primase complex can expand a template by a maximum of 40–90 nt. In a different study, longer products of a polyG:polyC duplex have been synthesized using the Klenow fragment of DNA Pol I (16). …”

Section: Introductionmentioning

confidence: 99%

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

Hartig

Kool

2005

Nucleic Acids Research

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Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.

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“…Synthesis of SH-poly(dG)-poly(dC)-SH was conducted as described in Materials and Methods and in our earlier publication [16]. Briefly, a double-stranded (ds) template primer, S-S-5'-(dG) 12 -(dC) 12 -5'-S-S, composed of 12 GC base pairs (bp) and containing disulfide groups at both 5' ends of the DNA, was extended by Klenow exo- fragments of Polymerase I in the presence of dGTP and dCTP.…”

Section: Resultsmentioning

confidence: 99%

“…Synthesis of 450 bp [S-S-5'-poly(dG)-3']-[3'-poly-(dC)-5'-S-S], the ds nucleic acid containing disulfide groups at both 5' ends of the molecule was performed as previously described [16,23] using Klenow exo- of DNA Polymerase I. A standard reaction mixture contained: 60 mM K-Pi (pH 7.5), 3.2 mM MgCl 2 , 5 mM DTT, 1 mM dCTP, 1 mM dGTP, 0.025 U/µL Klenow exo- and 2 µM of the template-primer.…”

Section: Methodsmentioning

confidence: 99%

Preparation, Characterization and Manipulation of Conjugates between Gold Nanoparticles and DNA

et al. 2016

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Here we described the preparation and characterization by atomic force microscopy of dumbbell-shaped conjugates between 450 bp double-stranded DNA polymer, poly(dG)-poly(dC), and 5 nm gold nanoparticles (GNPs). We have demonstrated that the size of the nanoparticles in the conjugates can be increased in a controlled fashion. Application of the conjugates for measuring the electrical conductivity of DNA is discussed.

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In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I

Cited by 57 publications

References 36 publications

Electronic structure of single DNA molecules resolved by transverse scanning tunnelling spectroscopy

Electronic structure of single DNA molecules resolved by transverse scanning tunnelling spectroscopy

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

Preparation, Characterization and Manipulation of Conjugates between Gold Nanoparticles and DNA

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