Light Cycler® qPCR
Real Time PCR with Light Cycler™ Hybridization Probes
The detection principle of LC™ Hybridization Probes (HybProbes) is Fluorescence Resonance Energy Transfer (FRET), the phenomenon of energy transfer from a donor to an acceptor fluorophore. If the donor and the acceptor fluorophore are in close proximity to each other, excitation of the donor by blue light results in energy transfer to the acceptor, which can then emit light of longer wavelength. This fact forms the basis for Roche’s real-time online LightCycler® PCR System. It allows formation of PCR products to be monitored by using two sequence specific, fluorescent labeled oligonucleotide probes, called Hybridization Probes, in addition to the PCR primers.
For this LC™ real-time PCR detection format the following are the major steps:
PCR template, primers and HybProbes are single-stranded. One HybProbe is labeled with the fluorescent donor dye (e.g. Fluo), the other one is labeled with one of the two available acceptor dyes (LCRed 640 or LCRed 705). The donor dye is excited by blue light of 470 nm and emits green light of 530 nm.
After reaching the annealing temperature, PCR primers and HybProbes hybridise to their specific target regions. The donor dye now comes into close proximity to the acceptor dye. Energy emitted from the donor dye excites the acceptor dye, which now emits red light of 640 or 705 nm.
After annealing to their target sites, the primers are elongated by thermostable DNA polymerase. Due to the increased temperature of 72°C during elongation, most HybProbes have already melted off. Probes that are still annealed to their target sequence are displaced by the protruding DNA polymerase.
The amount of template DNA has doubled and the DNA is double-stranded; HybProbes are displaced from their target site. The next cycle of PCR is ready to start again at step a).
HybProbes are designed as pairs. One probe is labeled with the donor (3'Fluo) and one with the acceptor (5' LCRed 640 or LCRed 705) dye. As FRET decreases with the sixth power of distance, HybProbes have to be designed to hybridise to adjacent regions of the template DNA (separated by 1-5 nucleotides). If both probes hybridise, the two dyes are brought close together and FRET to the acceptor dye results in a signal measurable by the built-in fluorimeter of the LightCycler®.
The fluorescence signal disappears by increasing temperature above the melting temperature of the oligos. This is due to the fact that the probes melt away from the template strand, which significantly increases the distance between the dyes.
Mismatches between the probes and the target decrease the melting temperature of the respective probe, compared to a perfectly matched probe. This effect can also be used to detect SNPs by melting curve analysis.
Selection of Primers and Primer-Probe Sets
1. In General and for Quantification Experiments
The successful performance of a specific and sensitive PCR experiment requires well designed PCR primers. This means that the primers should meet specifications like
- the target sequence should contain only a single annealing site
- the primers should not contain any intra-molecular sequence complementarities, which are prone to formation of secondary structures
- no inter-molecular complementarities should exist between the primers, which could potentially cause formation of primer-dimers
For the design of HybProbes all of the above listed criteria are crucial, too. In addition, probe sequences that could hybridise to the PCR primers should be avoided. Less important is the binding to a unique target sequence, as HybProbes only efficiently hybridise to the highly concentrated PCR product. Binding to the low concentration starting template will have little effect on signalling. Thus, probes have to be checked only against the PCR product sequence.
The strategy for selection of primer-probe sets should include the following analyses:
- Global sequence analysis (A)
Find regions of the target sequences most suitable for PCR.
- Thermodynamic analysis (B)
Match the Tm of primers and probes.
- Cross-complementarity analysis (C)
Minimize intermolecular interactions of primers and probes.
Before starting the search for primers and probes, we recommend that you check your target sequence for possible homologies with other sequences by applying a BLAST (Basic Local Alignment Search Tool) search. Public BLAST servers are available on the Internet (e.g. www.ncbi.nlm.nih.gov/BLAST/ or www.tigr.org or others). BLAST programs compare a query sequence to all the sequences in a specified database. Sequences are aligned in pair-wise fashion to identify regions of similarity. Using this method you can easily find sequences similar to the target sequence. To find specific primers and probes you should only use those target regions with minimum similarities to other sequences.
Subject of this analysis is the identification of target sequences that are free of motifs which might have a strong negative effect on the PCR. The following table describes these undesired sequence motifs as well as their potential negative effects on PCR:
|Global sequence analysis (A)|
|Direct repeats||A sequence of 4 or more nucleotides, which is repeated: (…TTAGCT…TTAGCT…)||Direct repeats may generate secondary binding sites for primers. Stable hybridisation to secondary binding sites results in non-productive binding of the primers to non-specific regions of the sequence. As a result, efficiency of DNA amplification and detection decreases. The worst case scenario gives you multiple amplicons from the same template!|
|Homopolymeric runs||A sequence of 4 or more identical nucleotides: (…TTTTTTTT…)||Homopolymeric runs can be considered a special case of direct repeats. Additionally, homopolymeric runs can cause ambiguous binding of oligos to their target site ("slippage effect").|
|Inverse repeats||A sequence motif of 4 or more nucleotides, which shows self-complementarity (stem loop or hairpin motifs): (…AATGGC….GCCATT…)||Due to competition between inter-molecular (primer-template, probe-target) hybridisation and intra-molecular hybridisation, inverse repeats can cause inefficient priming and probing of the target sequence. PCR reaction and/or probe binding can completely fail, due to formation of stable hairpins at the binding region, or inside the amplicon in general.|
Thermodynamic analysis should be done to match the melting temperatures of the primers and to pair them with the appropriate probes. To calculate the Tm of primers and probes we recommend you to use the unified nearest neighbor thermodynamics formula (metabion offers a biocalculator for this purpose. click here. This analysis is especially important to maximize the Tm shift between matched and mismatched probes in SNP detection experiments.
The following table should give you some guidelines for selection of suitable HybProbes:
|Thermodynamic analysis (B)|
|Guideline for HybProbe Tm||Comment|
|Probe Tm should be 5°C-10°C higher than primers' Tms||For successful generation of a fluorescence signal, both HybProbe oligonucleotides have to bind simultaneously to the ss target DNA during the annealing phase of the PCR. Given that primers are elongated by Taq Polymerase immediately after annealing even at temperatures below 72°C, this may result in early displacement of the probes by the enzyme. Eventually, this can also prevent the probe from binding, due to covering of the probe binding site by the newly synthesized DNA strand. Therefore, the probes’ Tm should be higher than the primers' Tm. This ensures strong binding of the probes during annealing and generates a signal before probe displacement by DNA polymerase occurs.|
|Probe binding should not be too stable (avoid a Tm >10° higher than primer Tm)||Extremely stable probes may interfere with the amplification step by preventing Taq polymerase from proceeding, which lowers the sensitivity of the assay.|
2. For Mutation Detection Experiments
HybProbes used for SNP analysis are called anchor and sensor probes. The one covering the polymorphism site should be the lowest melting probe and it is called “sensor probe”. The other oligonucleotide, called “anchor probe”, anneals to a part of the target sequence which is not polymorphic and adjacent to the sensor probe site. To guarantee that the sensor probe melts first, the Tm of the anchor probe should be about 5°C higher than that of the sensor probe.
Any base mismatch has a strong impact on the Tm of an oligonucleotide that binds to a target single strand. This is the reason for differences in melting temperatures between perfect probe-target matches (high Tm) and formed double strands containing one or more mismatches (lower Tm). The Tm shift depends on several factors including the nature of the mismatch and its neighboring base pairs.
During melting curve analysis, the temperature is increased slowly. If the sensor probe is perfectly matched to its target sequence, it will melt away at a relatively high temperature. If the sensor probe is mismatched, melting will occur at a lower temperature.
For the design of SNP detection primer-probe sets the target region of the sensor probe is already defined by the position of the SNP. The sensor probe is selected first by analysing the region surrounding the SNP for probe sequences matching the user defined Tm.
In a short summary, for the design of probes that are used to detect mutations, there are additional guidelines to be taken care of due to the specialized role of the two probes:
- Tm of anchor probe > Tm of sensor probe (about 5°C)
- Ideally position (predicted) mismatched base in the middle of the sensor probe; if a centre position is not possible, at least avoid the mismatch to be adjacent to the last two bases of the probe.
In general, two main (risk) factors are decisive for the success of your experiments on the LightCycler®- or any other (real time)-PCR System:
- The proper design of primers and/or probes and
- The quality of primers and/or probes in terms of purity and accuracy of sequence
metabion is at your service to turn these potential risk factors into success factors by
- supporting you in primer/probe design if needed
- supplying you with our metabion top quality oligonucleotides - primers as well as LC™ probes!
Even a perfectly designed oligo is worth nothing if the actual products you are working with are of poor quality. metabion's excellent expertise in oligo synthesis is shown in every part of production, from instrument technologies, to systems and protocols. Moreover, the outstanding knowledge and experience of our people will help you to achieve positive results and minimize failure risk. We are looking forward to a fruitful cooperation of mutual success!
Recommended Storage for LightCycler®-Oligonucleotides and other Fluorescent Probes
- Resuspend the probes to a concentration of 20 µM in 1x TE'.
- Aliquot 40 µl quantities of the solution into small dark colored Eppendorf tubes. Store at -20°C.
- For usage of the probes, take one of the frozen 40 µl aliquots (20 µM), thaw, and dilute to working stock concentration.
- Store this solution at 4 °C. Probes treated in that way will be good for one month.
Proper storage of any fluorescent probe increases its lifetime. In any case, it is recommended to test the probes immediately after receiving them.
When you receive your fluorescent-dye labeled probes, resuspend them to a concentration of 20 µM in 1x TE. Multiple freeze-thaw cycles should be avoided. Therefore, we recommend you to prepare 40 µl aliquots of the 20 µM probe stock (never store aliquots diluted to working concentration!!) in small dark colored Eppendorf tubes, and store them at -20°C.
Fluorescent-dye labeled probes with a concentration of 20 µM, stored at -20°C should be stable for one year. We recommend to label the tubes with preparation and/or expiration date.
For usage of the probes, take one of the frozen 40 µl aliquots (20 µM), thaw and dilute the probe to its working stock concentration (always shortly before using them!). Store at 4°C. Under these conditions the probe should be good for one month.
Be careful not to expose the probes to light for extended periods of time, e.g. when leaving them out on the lab bench, and always store in dark-colored tubes to reduce the degradative effects of light.
Legal Notices and Trademark Attribution
LightCycler Probes are sold under license from HoffmannLa-Roche Ltd. No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. Custom synthesis of LC probes and oligonucleotides containing LCRed dyes under licence of Roche Diagnostics. All products are for research only. LightCycler® is a trademark of Idaho Technology Inc.
Useful link: www.gene-quantification.info