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LightCycler® 2.0 Carousel-Based System Performance

 

Read in this article:
Speed
Dynamic Range
Sensitivity
Reproducibility

 

Speed

The design of the glass capillaries facilitates high speed thermal cycling. Their high surface-to-volume ratio permits extremely rapid thermal transfer, which minimizes formation of nonspecific products that can lead to an overestimate of copy numbers.

Together, the glass capillaries and the air-based temperature control of the LightCycler® 2.0 Instrument ensure rapid PCR. An entire 35-cycle run can be completed in as little as 30 minutes (with 20 µl capillaries) or 60 minutes (with 100 µl capillaries).

 

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Dynamic Range

For reliable quantification of a wide range of concentrations, the fluorescence detection unit must respond predictably to a wide dynamic range of signals. The LightCycler® Instruments can detect from 10 to 1010 copies in the same run. In this experiment, a recombinant plasmid containing the 438 bp amplicon of Chlamydia pneumoniae 16S rDNA was amplified.

Figure 1: Logarithm of the fluorescence detected during the amplification plotted against the cycle number. All of the samples, which contained from 10 to 1010 copies of the amplicon, produced clear signals.

 

 

 

 

 

 

 

 

Figure 2: Plot of the resulting crossing points (cycle numbers) versus logarithm of concentration. A linear relationship over the entire range of concentrations can be shown.

(Data provided by Dr. Udo Reischl, Institute of Medical Microbiology and Hygiene, University Hospital of Regensburg, Germany).

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Sensitivity

The sensitivity of a LightCycler® 2.0 Instrument experiment depends on several factors that are common to all conventional PCRs (i.e., the quality of template, primer design, reaction conditions).

Experiments have shown that, under optimal conditions, PCR can detect a single-copy gene in 3 pg of human genomic DNA (approximately one human genome equivalent). At this concentration, statistical considerations significantly affect assay sensitivity and reproducibility. According to a Poisson distribution, the probability that a target is actually present in a given sample is approximately 67%.

Other types of DNA, such as plasmid DNA, are detectable when 1 – 10 copies are present in a sample.

Figure 3:  Profile of a DNA amplification. The results show that the reaction can detect as little as one copy of the DNA.

 

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Reproducibility

The ability of a real-time PCR instrument to distinguish between different starting concentrations primarily depends on the quality of individual instrument components. The LightCycler® 2.0 Instrument can determine starting concentrations with very high resolution. Variability from sample to sample is extremely low. This outstanding reproducibility is primarily due to these instrument components:

  • continuously rotating sample carousel in a single thermal chamber, which ensures identical PCR conditions for every sample;
  • a single optical unit to ensure identical measurements.

Figure 4: Analysis of plasmid DNA dilutions that contained from 105 to 102 copies. The crossing point for 1000 copies showed a coefficient of variation (CV) of 0.15%; for 10,000 copies, the CV was 0.09%.