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Bio-Synthesis offers a complete array of real-time qPCR probes and primers in different combinations of quenchers and dyes ranging from the visible spectrum up to the near infrared range. We offer non-IP dyes such as 6-FAM, HEX and TET modification as well as exotic dyes. These probes and primers are synthesized at different scales using natural DNA base or RNA analogs such as Bridged Nucleic Acid (BNA) to improve techniques requiring high affinity or high specificity probes such as SNP detection, expression profiling and in situ hybridization.

With more than 30 years of experience in oligonucletoide synthesis, we have the expertise to design the most technically challenging probes and primers. Our scientists collaborate with researchers world-wide to develop novel base analogs and labels. We are able to meet customer specifications, no matter how complex. If you need help concerning real-time qPCR design or in selecting dye and quencher combination for use with a specific thermocycler, please do not hesitate to contact us.

qPCR probes are provided as below :

  • Multi-labeled and mimetic bases
  • Wide range of reporters and quenchers
  • DNA or BNA, chimeric mixed bases
  • Single or dual HPLC purified
  • QC by MALDI, analytical HPLC and Fluoroscan
  • Delivered lyophilized in tubes or other means as required

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 as SYBR® Green I binds non-specifically to double-stranded DNA. Upon binding, the dye undergoes a conformational change resulting in high fluorescent emission, allowing measurement of the total amount of double-stranded product present in the reaction 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 to improve assay sensitivity and specificity. BNA can be incorporated into any of qPCR probe 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 synthesize with any standard bases. BNA have greater thermal stability than conventional DNA or RNA and therefore form a stronger bond with the complementary sequences. The introduction of BNA chemistry into a qPCR probe may result in an increase in the 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.

We can label the dye at any position within an oligonucleotide. Certain modifications are not available for direct labeling at the time of synthesis, A primary amine modified oligonucleotide is used to covalently attach with dye via NHS conjugation chemistry; other cross-linking chemistries are also available depending on particular project specifications. Dual HPLC purification is used to remove failed sequences during the first HPLC purification. After labeling, a second HPLC is performed to remove unlabeled oligonucleotides and excessive dye in order to obtain a full length labeled oligonucleotide.

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

Ordering & Contact Information

  • Please contact us for additional information or send an email to info@biosyn.com.
  • You may also request an online quote.
  • To contact us by phone, please call 1-972-420-8505 or Fax at 1-972-420-0442
  • Orders may be placed using a purchase order (PO) or by credit card through our secure online ordering system.
  • When using a credit card ( creditcard ) it will be billed under "Bio-Synthesis, Inc."