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.
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LightCycler® 480 Detection Formats
| 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 |
Monitor PCR with the SYBR Green I Dye
Schematic diagram of SYBR Green I format
A During annealing, PCR primers hybridize to the target and form small regions of dsDNA where SYBR Green I intercalates; the fluorescent signal slightly increases.
B In the elongation phase, more dsDNA is formed and more SYBR Green I dye can intercalate; higher fluorescent signal
C At the end of the elongation phase, all DNA has become double-stranded and the maximum amount of SYBR Green I is intercalated. The fluorescence is measured (530 nm) at the end of each elongation phase.
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
A The donor-dye probe is labeled with fluorescein at the 3´ end and the acceptor-dye probe is labeled with LightCycler® Red at the 5´ end. Hybridization does not take place during the denaturation phase of PCR and, thus, the distance between the dyes is too large to allow energy transfer to occur
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
C After annealing, the temperature is raised and the HybProbe probe is displaced during elongation. At the end of this step, the PCR product is double-stranded and the displaced HybProbe probes are again too far apart to allow FRET to occur
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.
A The probe carries two fluorescent dyes in close proximity, with the quencher dye suppressing the reporter fluorescence signal. The 3´ end of the hydrolysis probe is phosphorylated, so it cannot be extended during PCR. During denaturation, the target double-stranded DNA is separated.
B In the annealing phase of PCR, primers and probes specifically anneal to the target sequence
C As the DNA polymerase extends the primer, it encounters the probe. The polymerase then cleaves the probe with its inherent 5´ nuclease activity, displaces the probe fragments from the target, and continues to polymerize the new amplicon
D In the cleaved probe, the reporter dye is no longer quenched and therefore can emit fluorescent light that can be measured by one channel of the LightCycler® optical unit. Thus, the increase in fluorescence from the reporter dye directly correlates to the accumulation of PCR products
SNP Genotyping and Mutation Detection with SimpleProbe Probes
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.
A During the denaturation phase, no hybridization takes place, thus, only a low fluorescence background is detected at 530 nm.
B During the annealing phase, the probe hybridizes to the amplified DNA fragment and is no longer quenched. Fluorescein, when excited by the LightCycler® LED, emits green fluorescent light which is measured only at the end of each annealing step at maximum intensity
C During the subsequent elongation step, the SimpleProbe probe is displaced
D At the end of the elongation step, the PCR product is double-stranded and the displaced SimpleProbe probe is again quenched.
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.
