Roche-LightCycler® 480 SYBR Green I Master（少量现货）

Save time with a convenient, ready-to-use 2x concentrated hot start master mix.

Minimize pipetting steps and contamination risk.

Perform sensitive, specific, and quantitative PCR or sequence detection using the LightCycler^® 480 Instrument.

Eliminate time-consuming MgCl₂ titration.

Achieve consistent, high quality performance with LightCycler^® 480 Instruments and Multiwell Plates.

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.

* 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

	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.

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

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

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.

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.

LightCycler^® 480 System Reagents

Read in this article:

General Concept
Benefits
Easy-to-Use One Component Master Mixes
Hot Start Enzymes for Optimized Specificity and Sensitivity
Carryover Prevention
Developed for or Readily Combined with RT-PCR
Compatibility
 

General Concept

The LightCycler^® 480 Real-Time PCR System includes generic master mixes tailor-made for each key application in genomics research (gene identification, gene quantification, variation screening, and genotyping).

Enzyme variants and buffer conditions have been carefully chosen and optimized for each application:

• LightCycler^® 480 SYBR Green I Master contains components which help minimize primer-dimers.
• LightCycler^® 480 Genotyping Master is optimized for melting curve analysis with HybProbe or SimpleProbe probes, especially in multiplex assays.
• LightCycler^® 480 High Resolution Melting Master, based on the saturating DNA dye ResoLight, allows heteroduplex analysis for mutation scanning and genotyping. LightCycler^® 480 RNA Master Hydrolysis Probes enables ultra-fast, sensitive one-step RT-PCR.

Benefits

• True, user-friendly one-component master mixes for exceptional sensitivity in fast PCR applications.
• Optimized shapes (slopes, heights) for amplification and melting curve, allowing bias-free Cp- and T_m-determination, respectively.
• Combination with highly reproducible LightCycler^® 480 thermal block cycler and optimized disposables eliminates the need for passive reference dyes.
• LightCycler^® 480 SYBR Green I Master and Probes Master available in pack sizes suitable for high-throughput applications.
• Extended stability at room temperature (see Figure 1) and at +4° to +8°C for up to four weeks, for added convenience with daily or routine use (LightCycler^® 480 SYBR Green I and LightCycler^® 480 Probes Master).

Figure 1: Stability of the LightCycler^® 480 SYBR Green I Master. PCR reactions of serial 1:10 dilutions (10,000–10 copies/reaction) with three replicates of a human DNA target sequence were set up. The amplification reactions were then run either immediately or after 24 hours incubation on a loading robot at room temperature. The amplification curves and the corresponding Cps of the directly-assayed (blue-colored curves) and delayed-assayed (red-colored curves) samples demonstrate that the performance of the hot start enzyme master mix was not affected by prolonged standing.

Easy-to-Use One Component Master Mixes

Since all mixes are provided as one-component master reagents, reaction setup requires only the addition of template DNA, primers, and except for experiments with SYBR Green I, probes.

The mixes can be used with different types of DNA (e.g., genomic, cDNA) and are ideally suited for high-throughput applications in 96- or 384-well plates.

Each master mix is optimized for a fixed MgCl₂ concentration, which works with nearly all primer combinations. No adjustment of the MgCl₂ concentration is needed to amplify different sequences.

Hot Start Enzymes for Optimized Specificity and Sensitivity

LightCycler^® 480 PCR master mixes are all based on enzyme compatible with hot start protocols. When used on the LightCycler^® 480 Instrument, these protocols have been shown to significantly improve the specificity, sensitivity, and yield of PCR. For example, heat-labile blocking groups on some of the amino acid residues of FastStart Taq DNA Polymerase make the modified enzyme inactive at room temperature. Therefore, there is no elongation during the period when primers can nonspecifically bind. The FastStart Taq DNA Polymerase is activated by removing the blocking groups at a high temperature (i.e., a pre-incubation step at +95°C).

Carryover Prevention

LightCycler^® 480 PCR master mixes contain a dNTP mix including UTP instead of dTTP. Therefore, they can be used in conjunction with heat-labile Uracil DNA Glycosylase for carryover prevention during PCR.

Developed for or Readily Combined with RT-PCR

LightCycler^® 480 master mixes can be used in two-step RT-PCR applications, for example, downstream of Transcriptor Reverse Transcriptase.

For one-step reactions, the LightCycler^® 480 RNA Master Hydrolysis Probes is available.

Compatibility

The LightCycler^® 480 master mixes have been adapted to the special rapid cycling environment of the LightCycler^® 480 Instrument and to the different probe chemistries supported by the system. Optimal experimental results can therefore only be obtained when the LightCycler^® 480 Instrument and reagents are used in combination. Due to the differences in design (dimensions, material) of the respective reaction devices, reagents are tailor-made and must not be interchanged between the LightCycler^® 480 and the classical, capillary-based LightCycler^® 2.0 and 1.5, or the new strip-based LightCycler^® Nano System.