Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions

@article{Higuchi1993KineticPA,
  title={Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions},
  author={Russell Higuchi and Carita Fockler and Gavin Dollinger and Robert Malcolm Watson},
  journal={Bio/Technology},
  year={1993},
  volume={11},
  pages={1026-1030}
}
We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting… 

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References

SHOWING 1-10 OF 18 REFERENCES

Simultaneous Amplification and Detection of Specific DNA Sequences

TLDR
The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample through–put.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

TLDR
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.

Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction.

TLDR
An adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells for expression of two cytokines and the copy number of the human GM-CSF gene in normal human cells is described.

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

TLDR
Improved amplification performance is evident for target copy numbers below approximately 10(3), and when mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization.

Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

TLDR
The effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR) were investigated, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches.

Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants.

TLDR
The results suggest that non-isotopic PCR methods are sensitive, specific, and potentially useful in the early diagnosis of HIV-1 infection in developed and developing countries.

Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2).

TLDR
The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS), was determined and the cloned gag and env genes of ARv-2 were shown to express viral proteins.

High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR.

TLDR
Quantitation of HIV-1 in plasma by QC-PCR may be useful in assessing the efficacy of antiretroviral agents, especially in early stage disease when conventional viral markers are often negative.