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… Expand
Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes
TLDR
This chapter discusses the various approaches to monitor the PCR amplifications including cycle-by-cycle monitoring and empathizes on the use of a charged-coupled device (CCD) camera, which is a simpler, less expensive method of monitoring multiple PCRs. Expand
Real-Time Polymerase Chain Reaction
TLDR
This chapter provides an overview of the real-time polymerase chain reaction (PCR) methodology and its applications, and numerous adaptations and applications to this classic end-point PCR are described, which include semiquantitative PCR, quantitative competitive PCR, and real- time PCR. Expand
Doubling Throughput of a Real-Time PCR
TLDR
A method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening and can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel. Expand
Real-Time Polymerase Chain Reaction (PCR) Quantitation of DNA
TLDR
In this laboratory experiment, real-time PCR will be conducted using serially diluted standard K562 DNA, published human TPOX single locus DNA primers, and Bio-Rad’s iQ SYBR Green Supermix employingSYBR Green I to amplify the 64-base pair amplicon. Expand
Real-Time PCR – The Basic Principles
Conventional PCR is a powerful technique that allows exponential amplification of DNA sequences. A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers areExpand
Quantitative PCR Techniques
The polymerase chain reaction (PCR) is a primer-mediated, enzymatic amplification of specific genomic DNA or cDNA sequences. RNA-based assays for transcript quantification are arranged vertically,Expand
Application of Real-time Polymerase Chain Reaction (RT-PCR)
TLDR
RT-PCR could be combined with reverse transcription polymerase chain reaction to quantify messenger RNA (mRNA) at a particular time for in a particular cell or tissue type. Expand
Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes.
TLDR
Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol. Expand
Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.
TLDR
Simple DNA extension rate assays can be performed on real-time PCR instruments and 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. Expand
In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements.
TLDR
The total number of cycles and total time required to reach maximum fluorescence is reduced as compared to conventional PCR and the extension time of each PCR cycle can be individually optimized while the reaction is in progress. Expand
...
1
2
3
4
5
...

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. Expand
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. Expand
Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.
TLDR
An alternative method for the synthesis of specific DNA sequences is explored that involves the reciprocal interaction of two oligonucleotides and the DNA polymerase extension products whose synthesis they prime, when they are hybridized to different strands of a DNA template in a relative orientation such that their extension products overlap. Expand
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. Expand
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. Expand
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 detectableExpand
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. Expand
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. Expand
Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products.
TLDR
Analysis of ethidium-bromide-stained RT-PCR products can be used to provide a "semiquantitative" indication of relative levels of specific transcripts in a developing cell lineage without using radioactive probes to quantitate these products, indicating a coordinate inactivation of the X-linked loci at the onset of meiosis. Expand
Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.
TLDR
The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction and DNA extracted from bloodstain seems less prone to contain PCR inhibitors when prepared by this method. Expand
...
1
2
...