LightCycler^® 96 System Performance Data

Real-time PCR was done using 30 ng of human DNA in each of the 96 block positions. (A) A 110 bp amplicon of the beta globin gene was amplified using SYBR Green I detection. (B) The reaction product was also subjected to melting curve analysis. In summary, low variation Cq values (Cq range = 0.16 and SD = 0.033) and overlapping melt curves of the amplicon (TM range of 0.28°C and SD = 0.063) in each of the 96 positions demonstrated temperature homogeneity and equal treatment of all samples - independent of block position.

A Parvo B19 gene fragment was amplified in ten serial 1:10 dilutions ranging from 109 to 100 copies per well, and detected with Universal ProbeLibrary (UPL) Probe #137. Ten replicates were run for each dilution (only 9 for the 4 highest concentrations). (A) Results on the LighCycler® 96 System show excellent reproducibility and resolution down to very low copy numbers. (B) On a competitor instrument, Cq standard deviation values for a given concentration were much higher and Cq differences between dilution steps varied more across the whole dilution range.

The LightCycler® 96 Software’s Qualitative Detection Module allows for reliable analysis of target genes and controls, using two or even more colors. By using an internal control (IC) with the target in the same or a different well, inhibition of the PCR reaction (e.g., impurities originating from sample preparation) can be monitored.

The internal control can be either an endogenous target gene (ß-globin in humans) or an exogenous target gene (viral nucleic acid or synthetic target). For easy analysis, the LightCycler® 96 qualitative detection module includes a control concept: based on the individual calls for target gene(s) and internal control, a combined call is automatically generated and the overall result visualized via an intuitive heatmap.

The Qualitative Detection Module provides a Combined Call by combining the individual calls of a target gene and an internal control (IC).
Three basic result types are reported:

1. [Green] Positive combined call: target call positive, IC call positive or negative (e.g., wells A2, A3, B2; positions labeled 1, 2, 4)
2. [Red] Negative combined call: Target call negative, IC call positive (e.g., well F2; positions labeled 6)
3. [Yellow] Invalid combined call: Target call negative, IC call negative (e.g., well G2; positions labeled 7)

High Resolution Melting (HRM) is a homogeneous, closed-tube, post-PCR technique enabling rapidly and efficient discovery of genetic variations (e.g., SNPs, insertions, deletions, methylated regions).

In LightCycler® HRM experiments, sample DNA is first amplified in the presence of ResoLight, a special type of saturating DNA dye contained in the LightCycler® 480 High Resolution Melting Master.

Using the instrument’s high data acquisition rate, a melting curve is generated, and the resulting data is analyzed in four steps (see Figure, left part). Signal differences between each curve and a chosen reference are plotted, allowing the automatic clustering of samples into distinct groups that have similar melting curve shapes (e.g., heterozygotes versus homozygotes).

High Resolution Melting on the LightCycler® 96 System. Left part: A 72 bp fragment of the HFE gene including a class III SNP was amplified from 10 ng of human genomic DNA using the LightCycler® 480 High Resolution Melting Master containing ResoLight dye. Analysis of generated HRM data was done in four steps: A: original melting curves, B: normalized melting curves, C: normalized melting peaks, D: difference plot. Right part: Two different competitor instruments tested with the same assay failed in detecting all present groups of sequence variation (green group seen in D is missing in E and F).

Notably, with the LightCycler® 96 Software’s HRM module,

• analyses are always generated in a gene-specific manner (e.g., when multiple genes are contained in different wells of the same run).
• an automated algorithm calculates groups based on automated normalization slider and sensitivity settings.
• the "Normalized Melting Peaks" chart enables improved discrimination of complex groupings compared, for example, to the difference plot. (see example with 6 genotypes of TNF alpha in Figure, C)
• automatic and manual annotation are supported heat maps are provided as an additional option for result visualization (see Figure, D)

HRM analysis of a 111 bp fragment of the human TNF alpha gene present in six different sequence variants. Human genomic DNA isolated from different donor samples was used as template in technical replicates. PCR was done using the LightCycler® 480 High Resolution Melting Master, including ResoLight dye for detection and subsequent high resolution melting analysis. A: original melting curves, B: normalized melting curves, C: normalized melting peaks, D: color-coded overview of observed variants.