Real-time monitoring of DNA polymerase activity using molecular beacon

2006 ◽  
Vol 353 (1) ◽  
pp. 141-143 ◽  
Author(s):  
Changbei Ma ◽  
Zhiwen Tang ◽  
Kemin Wang ◽  
Weihong Tan ◽  
Jun Li ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Molecular Beacon ◽  
Polymerase Activity ◽  
Real Time Monitoring

Label-free molecular beacon for real-time monitoring of DNA polymerase activity

2016 ◽  
Vol 408 (12) ◽  
pp. 3275-3280 ◽  
Author(s):  
Changbei Ma ◽  
Haisheng Liu ◽  
Jun Wang ◽  
Shunxin Jin ◽  
Kemin Wang
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Molecular Beacon ◽  
Polymerase Activity ◽  
Label Free ◽  
Real Time Monitoring

scholarly journals Real-time monitoring of DNA immobilization and detection of DNA polymerase activity by a microfluidic nanoplasmonic platform

2019 ◽  
Vol 142 ◽  
pp. 111528 ◽  
Author(s):  
Johanna Roether ◽  
Kang-Yu Chu ◽  
Norbert Willenbacher ◽  
Amy Q. Shen ◽  
Nikhil Bhalla
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Real Time Monitoring ◽  
Dna Immobilization

scholarly journals Influence of PCR Reagents on DNA Polymerase Extension Rates Measured on Real-Time PCR Instruments

2014 ◽  
Vol 60 (2) ◽  
pp. 334-340 ◽  
Author(s):  
Jesse L Montgomery ◽  
Carl T Wittwer
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Dna Polymerase ◽  
Real Time Pcr ◽  
Monovalent Cation ◽  
Polymerase Activity ◽  
Simple Extension ◽  
Extension Rate ◽  
Nucleotide Analogs ◽  
Dna Dyes

Abstract BACKGROUND Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. METHODS A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30–90 min. RESULTS The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1–100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%–90% activated after 20 min. CONCLUSIONS Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

scholarly journals Real-Time Monitoring of DNA Polymerase Function and Stepwise Single-Nucleotide DNA Strand Translocation through a Protein Nanopore

2010 ◽  
Vol 122 (52) ◽  
pp. 10304-10307 ◽  
Author(s):  
John Chu ◽  
Marcos González-López ◽  
Scott L. Cockroft ◽  
Manuel Amorin ◽  
M. Reza Ghadiri
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Real Time Monitoring ◽  
Single Nucleotide ◽  
Dna Strand

scholarly journals Real-Time Monitoring of DNA Polymerase Function and Stepwise Single-Nucleotide DNA Strand Translocation through a Protein Nanopore

2010 ◽  
Vol 49 (52) ◽  
pp. 10106-10109 ◽  
Author(s):  
John Chu ◽  
Marcos González-López ◽  
Scott L. Cockroft ◽  
Manuel Amorin ◽  
M. Reza Ghadiri
Keyword(s):  
Real Time ◽  
Dna Polymerase ◽  
Real Time Monitoring ◽  
Single Nucleotide ◽  
Dna Strand

Real time monitoring of nucleic acids ligation based on molecular beacon

2003 ◽  
Vol 48 (12) ◽  
pp. 1215-1218 ◽  
Author(s):  
Zhiwen Tang ◽  
Kemin Wang ◽  
Weihong Tan ◽  
Jun Li ◽  
Lingfeng Liu ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Real Time ◽  
Molecular Beacon ◽  
Real Time Monitoring ◽  
Nucleic Acids Ligation
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