Quantitative real-time PCR (qPCR) relies on real-time detection of amplification products as it is accumulated during PCR. The progress of the reaction is monitored by tracking the increase in fluorescence from an associated reporter molecule to detect the concentration of target. The use of non-specific DNA binding dyes or sequence-specific probes are two fluorescent tracking systems use in qPCR applications.
Probe-based Real-time qPCR Detection
Probe-based detection methods rely on one or more fluorescently-labeled oligonucleotides that are positioned between the two PCR primers. Because the probe is sequence specific, it will only detect the presence of a single amplicon within the reaction.
There are several types of probe design that can be used including:
- Dual-Labeled Probes
- Molecular Beacons
- LightCycler Probes, Hybridization FRET probes
- Scorpions Probes
- Plaxor Probes
Each probe type enables researchers to measure an increase in fluorescent signal that corresponds to an increase in the copy number of the desired amplicon. In addition to the increase in sensitivity that is gained from using sequence-specific probes for detection, these probes can also be labeled with different fluorescent dyes, allowing detection of multiple targets within the same PCR reaction.
- High specificity
- Sensitive to low # of copies, ~1-10 copies
- High reproducibility
- Applicable in Multiplexing or pre-designed assays
- High level of quantitation in gene expression
Applications used in probe-based qPCR detection include:
- Gene expression
- DNA quantitation
- CHiP
- SNP genotyping
- Copy number variation
- Pathway analysis
- microRNA & small RNAs
- Mutation detection
- Protein analysis
- Multiplexing
Non-specific DNA Binding Dye Detection
DNA binding dye, such as SYBR® Green I, binds non-specifically to double stranded DNA. Upon binding, the dye undergoes a conformation change, resulting in high fluorescent emission. This emission allows measurement of the total amount of the double-stranded product present after each amplification cycle. SYBR® Green-based detection provides the following:
- Low cost and ease of use
- Medium specificity
- Variable sensitivity for low # of copies
- Medium reproducibility
- Not applicable for Multiplexing
- Low level quantitation for gene expression
Applications using SYBR system
- Gene expression
- DNA quantitation (pathogen detection)
- CHiP
qPCR Primer Design
Proper optimization can increase the efficiency and sensitivity of a qPCR reaction.
Primer and Probe Design
Average length of a qPCR probe is about 25-35 bases, please be aware that sequences longer than 30 bases are unlikely to have efficient quenching as an end-labeled probe. For such situations, please consider positioning the quencher internally instead. Conversely, sequences shorter than 20 bases may have been designed as a BNA probe, improving assay sensitivity and specificity. BNA can be incorporated into any qPCR probes and primer types, providing enhanced sensitivity and specificity for your assays. BNA is a novel nucleic acid analog containing a 2’-O,4’- aminoethylene six-member bridge. Probes or primers can be synthesized with any standard base. BNA has greater thermal stability than conventional DNA or RNA, forming a stronger bond with the complementary sequences. The introduction of BNA chemistry into a qPCR probe may result in an increase in duplex melting temperature (T M) of up to 8 °C per BNA monomer substitution in medium salt conditions, compared to a DNA fluorescent probe. It is possible to optimize the TM level and the hybridization specificity through specific placement of the BNA base(s) in the probe design.
Contact us for BNA qPCR Primer probe design.
Primer and Probe Placement
In general, amplicons should be between 50-200 bases in length. Shorter amplicons tend to be more tolerant of less than ideal reaction conditions, improving the consistency of results.When quantifying RNA targets, select primers spanning exon-exon junctions to avoid amplification of contaminating genomic DNA in cDNA samples.
Primer and Probe Concentration
Optimization of primer and probe concentrations can improve the detection level of a particular amplicon by around 10 cycles depending on the sequence. Since a 3 Cq difference in amplicon detection indicates approximately a 10 fold difference in template concentration, this simple step can greatly improve both the accuracy and sensitivity of the reaction.
Buffer Conditions
Assay performance may also benefit from optimization of buffer components (particularly MgCl2) and the internal reference dye. Optimizing the concentration of these components is especially important when designing multiplex assays or singleplex assays in which design of an appropriate probe/primer combination proves to be difficult.
Specifications
Quality control:
Every oligo is quality checked by MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), capillary electrophoresis (CE), and/or polyacrylamide gel electrophoresis (PAGE).
Purification
Fully deprotected and desalted. Purified by PAGE or RP-HPLC
Length
|
Length |
| LightCycler probes |
15 to 40 mers (optimal length: 20 to 30 mers) |
| Dual-labeled fluorogenic probes: |
15 to 40 mers |
| Molecular Beacons |
15 to 40 mers |
| Scorpions probes |
30 to 60 mers (uni-probe) |
| 15 to 45 mers (bi-probe) |
| Plexor |
up to 30 mers |
Backbone
Phosphodiester bond
Format
Delivered as dried-down product in opaque tubes
Turn-around time:
|
Turn-around time |
| LightCycler probes |
4 to 5 working days |
| Dual-labeled fluorogenic probes: |
5 to 6 working days |
| Molecular Beacons |
5 to 6 working days |
| Scorpions probes |
7 to 10 working days |
Turn-around time is dependent upon successful QC validation and does not include shipping time.
Storage and stability:
See information on our oligos: storage recommendations
Shipment:
Shipped by mail or express delivery, dry, in individual, opaque tubes
Technical Documentation:
Oligonucleotides are delivered with an OligonucleotideTechnical Data Sheet, which includes oligonucleotide name, sequence, concentration, precise quantity in OD and nmols, Tm, MW, length, extinction coefficient and purification data.
Additional Services:
- Probe design services
- High-throughput screening formats (microplates, etc)
- Aliquoting
Additional services may increase turn-around time
Pricing:
Please contact your local Bio-Synthesis representative
Ordering:
On-line (contact us), by email (info@biosyn.com) or fax