LightCycler® 480 Detection Formats
Due to its highly flexible selection of excitation and emission wavelengths, the LightCycler® 480 Real-Time PCR System is compatible with most currently available dyes and assay formats, yet is also adaptable to new, future applications.
The following assay formats can be used on the LightCycler® 480 Real-Time PCR System:
- Universal ProbeLibrary
- SYBR Green I
- HybProbe Probes
- Hydrolysis Probes
- SimpleProbe Probes
- High Resolution Melting (HRM)
A summary of LightCycler® 480 Real-Time PCR System detection formats is provided in the table below.
Applications | Assay Detection Format | Dyes | Excitation (nm) | Detection (nm) |
Product Characterization | SYBR Green l | SYBR Green I | 465 | 510 |
Quantification | Monocolor Hydrolysis Probe / UPL Probe |
FAM | 465 | 510 |
Dual Color* Hydrolysis Probes / UPL Probes |
FAM VIC/Hex/Yellow 555 |
465 533 |
510 580 |
|
3 Colors Hydrolysis Probes |
FAM VIC/Hex Cy 5/Cy 5.5 |
465 533 618 |
510 580 660 |
|
4 Colors Hydrolysis Probes |
LightCycler® Cyan 500 FAM LightCycler® Red 610 Cy 5/Cy 5.5 |
440 498 533 618 |
488 580 610 660 |
|
Melting Curve Genotyping (SNP Analysis) |
Monocolor HybProbe Probes |
Fluorescein (Donor) LightCycler® Red 640 |
498 | 640 |
Monocolor HybProbe Probes |
Fluorescein (Donor) LightCycler® Red 640 |
498 | 640 | |
Multicolor HybProbe Probes |
Fluorescein (Donor) LightCycler® Red 610 LightCycler® Red 640 Cy 5/Cy 5.5 |
465 498 498 498 |
510 610 640 660 |
|
Endpoint Genotyping (SNP Analysis) |
Dual Color* Hydrolysis Probes |
FAM VIC |
465 533 |
510 580 |
Melting Curve Genotyping (SNP Analysis) |
SimpleProbe Probes | Fluorescein | 465 | 510 |
Gene Scanning, Mutation Discovery, and Methylation Analysis | High Resolution Melting | HRM Dye | 465 | 510 |
* Universal color compensation objects for dual color FAM/VIC and UPL assays.
Figure 1: LightCycler® 480 excitation and emission filters at a glance.
Monitor PCR with the SYBR Green I Dye
When the SYBR Green I dye intercalates into dsDNA, its fluorescence increases greatly. During the different stages of PCR, the intensity of the fluorescent signal will vary, depending on the amount of dsDNA present.
Schematic diagram of SYBR Green I format
Monitor PCR with the LightCycler® HybProbe Format
TheLightCycler® HybProbe format is based on fluorescence resonance energy transfer (FRET). Two sequence-specific oligonucleotide probes are labeled with different dyes (donor and acceptor), and are added to the reaction mix along with the PCR primers. During the annealing phase, HybProbe probes hybridize to the target sequences on the amplified DNA fragment in a head-to-tail arrangement, thereby bringing the two dyes close to each other. The donor dye (fluorescein) is excited by the blue LED. As long as the two dyes are close to each other (within 15 nucleotides), the energy emitted by the donor dye excites the acceptor dye on the second HybProbe probe, which then emits fluorescent light at a different wavelength. This fluorescence is directly proportional to the amount of target DNA generated during PCR. HybProbe probes are displaced during the elongation and denaturation steps.
Advantages of the HybProbe Format
- Only the presence of a specific amplification product causes an increase in fluorescence.
- Increased specificity because two sequence-specific probes hybridize to the target.
- Primer-dimers do not interfere because they are not recognized by the sequence-specific probes.
- Probe sequences are not altered by PCR, so they can still be used in a subsequent assay, such as for mutation detection or SNP analysis

B During the annealing phase, the probes hybridize to the amplified DNA fragment in a close head-to-tail arrangement. When fluorescein is excited by the light from the LED, it emits green fluorescent light, transferring the energy to LightCycler® Red, which then emits red fluorescent light. This red fluorescence is measured at the end of each annealing step, when the fluorescence intensity is highest
Monitor PCR with Hydrolysis Probes
Hydrolysis probe assays are actually homogenous 5´ nuclease assays, since a single 3´ non-extendable hydrolysis probe, which is cleaved during PCR amplification, is used to detect the accumulation of a specific target DNA sequence. This single probe contains two labels, a fluorescence reporter and a fluorescence quencher, which are close to each other.
When the probe is intact, the quencher dye is close enough to the reporter dye to suppress the reporter fluorescent signal (via FRET). During PCR, the 5´ nuclease activity of the polymerase cleaves the hydrolysis probe, separating the reporter and quencher. In the cleaved probe, the reporter is no longer quenched and emits a fluorescence signal.
SimpleProbe probes are a special type of hybridization probes. SimpleProbe assays differ from HybProbe assays in one important way: each assay requires only a single probe. This probe hybridizes specifically to a target sequence that contains the SNP of interest. Once hybridized, the SimpleProbe probe emits a greater fluorescent signal than it does when it is not hybridized to its target. Thus, changes in fluorescent signal depend solely on the hybridization status of the probe.
SimpleProbe probes are an excellent tool for SNP genotyping and mutation detection because they readily identify wild type, mutant, and heterozygous samples, yet are as simple to design and use as standard PCR primers.
High Resolution Melting (HRM) Analysis with LightCycler® 480 ResoLight Dye
LightCycler® 480 ResoLight Dye belongs to a new generation of dsDNA-binding dyes that can detect the presence of heteroduplexes formed during PCR (e.g., if the sample is heterozygous for a particular mutation). Its possible applications therefore exceed those of other, more traditional DNA-staining dyes, such as SYBR Green I. LightCycler® 480 ResoLight Dye is not toxic to amplification enzymes. Thus, high concentrations of the dye do not affect the PCR. These high concentrations completely saturate the dsDNA in the sample. Therefore, when dye molecules dissociate from dsDNA during melting, there is only little chance for them to re-bind to other unoccupied sites. This makes the melting process highly homogeneous and the acquired signals very sharp. Under these conditions, even small changes in the melting curve result in subtle, but reproducible changes in fluorescence.
For more information on HRM analysis and application examples, click here.