vent  (New England Biolabs)

Journal: BMC Microbiology

Article Title: Large scale multiplex PCR improves pathogen detection by DNA microarrays

doi: 10.1186/1471-2180-9-1

Figure Lengend Snippet: Large scale multiplex PCR with 800 primer pairs . Gel electrophoresis of PCR products obtained with high complexity 800-primer pair mix (Additional file 1 ) with a final concentration of 0.02 μM for each individual primer pair and using Taq polymerase (standard LSplex) (A) or using vent exo-polymerase (B and C). Efficiency of LSplex using primer mix with different individual primer concentrations (B). Optimized LSplex amplification of various DNA templates from Gram-negative, Gram-positive bacteria and Candida albicans (C). 100 ng genomic DNA from each indicated species served as template.

Article Snippet: Optimized LSplex using Vent exo- DNA polymerase amplification reactions contained 1× ThermoPolBuffer (New England Biolabs), 4 mM MgCl2 , and 0.2 mM of dATP, dCTP, dGTP, and dTTP (Sigma).

Techniques: Multiplex Assay, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Concentration Assay, Amplification

Journal: ACS Sensors

Article Title: Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits

doi: 10.1021/acssensors.0c00593

Figure Lengend Snippet: Tetrastable system built from two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA-sensing circuits (cT, aT, pT, and rT) are interconnected by kT αkβ and βkα, which repress unwanted cross-activation. (b) Detailed mechanism of the five kinds of templates (pol. = Vent(exo-), nick. 1 = Nt.BstNBI, nick. 2 = Nb.BsmI, RE = BsmI, and exo. = ttRecJ). cTs convert the complementary microRNA target to a signal strand (α or β). Autocataytic templates (aTs) exponentially amplify the signal strands. pTs, by deactivating a fraction of signal strands, suppress background amplification stemming from biochemical noise. Reporting templates (rTs) transduce the molecular signal (α or β) to a detectable fluorescence signal (green = Oregon green fluorophore, red = Atto633 fluorophore). From the α or β strands, killer templates (kTs) produce pTs of the opposite switch, mitigating unspecific cross-talks. All produced strands are continuously degraded by the exonuclease to maintain the system dynamics and avoid system poisoning by the accumulation of DNA strands. Only one half of the tetrastable circuit is represented here, the second half being obtained by substituting α by β and conversely.

Article Snippet: The nicking enzymes Nb.BsmI and Nt.bstNBI, the restriction enzyme BsmI, the DNA polymerase Vent(exo-), BSA, and dNTP were obtained from New England Biolabs (NEB).

Techniques: Activation Assay, Amplification, Fluorescence, Produced

Journal: Nucleic Acids Research

Article Title: Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand

doi: 10.1093/nar/gkr704

Figure Lengend Snippet: Schematic representation of PADDA and identified DNA damage. ( A ) A single non-cycled primer extension performed with a 5′-biotin-tagged primer and Vent exo– DNA polymerase identifies damaged nucleotides (inverted triangles), and generates a pool of highly specific biotin-tagged extended products (EP), each of them derived from a single DNA molecule and strand. Each extended product has a stop (indicated by a red star), which represents replicative arrest by a damaged nucleotide, a nick or random polymerase stalling. Some extended products will contain misincorporations (indicated by a solid red circle) that represent polymerase lesion by-pass with misincorporation. After several purification steps that include the use of biotin-binding, streptavidin-coated, paramagnetic beads, the strand-specific, highly purified, biotin-bound extended products can be used for damage fingerprinting or quantification. ( B ) For strand specific fingerprinting analysis (f-PADDA) of damage and repair at the single-nucleotide level, the purified pool of EPs is ligated to a chemically modified oligonucleotide-adapter using a highly efficient single-strand DNA ligase. The pool of adapter-linked EPs can be diluted to one EP per well and each EP is directly amplified and sequenced, or the EPs can be amplified as a pool, cloned and sequenced. In either case high fidelity amplification is performed using an oligonucleotide complementary to the adapter–primer and a nested gene-specific primer. Data is obtained in

Article Snippet: Vent exo–, a truncated form of a native DNA polymerase (Vent, NEB), was chosen for this assay instead of the currently used Sequenase ( ) or Taq DNA polymerase , because of its reported high sensitivity to detect DNA damage ( ) and primer-extension efficiency ( ).

Techniques: Derivative Assay, Purification, Binding Assay, Modification, Amplification, Clone Assay

Journal: Chembiochem : a European journal of chemical biology

Article Title: Polymerase Amplification, Cloning, and Gene Expression of Benzo-homologous "yDNA" Base Pairs

doi: 10.1002/cbic.200800339

Figure Lengend Snippet: Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis DNA polymerase (Vent exo−) and Klenow fragment of DNA

Article Snippet: The enzymes used were the exonuclease-deficient Klenow fragment of DNA polymerase I (Cat. No. M0212L from New England Biolabs) and the exonuclease-deficient Vent DNA polymerase from Thermococcus litoralis (Cat. No. M0257S from New England Biolabs.

Techniques: