High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer

Heng, Yang; Jiang, Hujun; Fang, Wei-Yi; Xu, Yuan; Zhang, Jia; Liao, Duan‐Fang; He, Fuchu

doi:10.1016/j.bbrc.2004.12.159

Cited by 19 publications

(9 citation statements)

References 21 publications

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“…3b). This probably might be due to the 3 0 ?5 0 exonuclease activity of the proofreading polymerase enzyme used in DNA amplification as previously reported (de Noronha and Mullins 1992;Wang et al 2000;Yang et al 2005).…”

Section: Distinguishing T Weissflogiimentioning

confidence: 99%

Development of primers and a procedure for specific identification of the diatom Thalassiosira weissflogii

et al. 2010

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A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3 0 -end of 18S rDNA through 5 0 -end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.

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Section: Distinguishing T Weissflogiimentioning

confidence: 99%

Development of primers and a procedure for specific identification of the diatom Thalassiosira weissflogii

et al. 2010

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“…Such modifications did not alter the stability of lengthy DNA–complexes, but their presence increased the discrimination of nucleotide mismatches, single or multiplex, and even those located at a distance from the enzymatic conversion site. The DNA–polymerases showed no detectable elongation of the modified oligonucleotide, even when the mismatches were located up to 8 nucleotides from the 3’–terminus of the primer [111]. Notably, according to the data presented above, DNA–polymerases form tight interactions with the carbohydrate–phosphate backbone of the DNA helix up to the above–mentioned position in the primer strand.…”

Section: Modifications Of the Internucleotide Phosphodiester Residuementioning

confidence: 99%

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DN

Vinogradova

Пышный

2010

Acta Naturae

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The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA–dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA–dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA–dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme–substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA–substrates. We also discuss relevant approaches to increasing the selectivity of enzyme–dependent reactions. These approaches involve the use of modified oligonucleotide probes which “disturb” the native structure of the DNA–substrate complexes.

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“…Total RNA from normal ECV304 cells, and those cells transfected by CAV-1-shRNA pENTR/U6 entry plasmid, CAV-1-pLenti6/BLOCK-iT lentiviral expression plasmid and S100A13 RNAi lentiviral plasmid was extracted using Trizol, and reversely transcribed into cDNA following the instructions of the SuperScript III cDNA reverse transcription kit. The solution containing 10 6 copies of pcDNA3.1 CAV-1 expression plasmid was diluted at five multiple proportionally and used as a template to draw a standard curve [10,11]. SYBR green I (0.5 µl) was added into 25 µl PCR reaction, denaturated at 94 ºC for 45 s, renaturated at 55 ºC for 30 s, and extended at 72 ºC for 40s, 30 cycles.…”

Section: Real-time Quantitative Pcr Validated Rnai Efficiencymentioning

confidence: 99%

Small Interfering RNA-mediated Caveolin-1 Knockout on Plasminogen Activator Inhibitor-1 Expression in Insulin-stimulated Human Vascular Endothelial Cells

Yang

Quan

et al. 2007

ABBS

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Using human vascular endothelial cells (ECV304) as the target, we studied the effect of caveolin (CAV)-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor (PAI)-1. The appropriate single-stranded oligonucleotides representing the RNAi CAV-1 gene were analyzed by Ambion software. After annealing to generate double-stranded oligonucleotides (ds oligo), it was cloned into the pENTR/U6 entry vector containing RNA polymerase III expression element by T4 DNA ligase. The short hairpin (shRNA) sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iT-DEST vector with an LR recombination reaction. After identification by sequencing, we successfully constructed the CAV-1 RNAi lentiviral expression system using Gateway technology. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blotting. ECV304 cells were cultured in the medium containing different concentrations of insulin (1x10(-9) to 1x10(-7) M) with the CAV-1 gene silenced or not. The expression level and subcellular localization of PAI-1 and CAV-1 were compared using reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blot assay. The results showed that the potent inhibition of CAV-1 expression could reach 85%, and it was specific to the CAV-1-derived shRNA, not the S100A13-derived shRNA. There was no dramatic difference in PAI-1 expression between the RNAi+ and RNAi- ECV304 cells incubated with physiological insulin, but PAI-1 protein did accumulate under the cell membrane. As the concentration of insulin increased, the expression of PAI-1 was up-regulated, whereas the expression of CAV-1 attenuated. Furthermore, PAI-1 clearly augmented after CAV-1 knockdown. These results indicated that hyperinsulinism could promote PAI-1 expression by inhibiting CAV-1, and stabilizing or up-regulating CAV-1 expression in endothelial cells might reduce complications of the great vessels and capillary vessels in diabetes.

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High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer

Cited by 19 publications

References 21 publications

Development of primers and a procedure for specific identification of the diatom Thalassiosira weissflogii

Development of primers and a procedure for specific identification of the diatom Thalassiosira weissflogii

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DN

Small Interfering RNA-mediated Caveolin-1 Knockout on Plasminogen Activator Inhibitor-1 Expression in Insulin-stimulated Human Vascular Endothelial Cells

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