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Custom qPCR and dPCR Probes

Custom qPCR and dPCR Probes
 
 

Increase the sensitivity and specificity of your real-time or digital PCR with probes from Microsynth. High-quality qPCR and dPCR probes with a broad range of dyes and quenchers are delivered at exceptional speed.

 

Features and Benefits

 
 
Highest Quality
  • Thorough purification processes (HPLC or even PAGE)
  • Stringent quality control (online trityl monitoring and MALDI-TOF MS)
 
Very Fast Production Times
  • within 3 to 5 working days
 

User-friendly Online Ordering System

  • Easy-to-use online portal with a series of helpful tools (e.g. order tracking & history, convenient search and re-order option)

 
 
Comprehensive Service Portfolio
  • Possibility to adjust binding affinity via MGB, LNA and other Tm enhancers or even combine different Tm enhancers to tailor your assay
  • Wide variety of fluorophores - quencher combinations
  • Professional design service
  • EN ISO 13485:2016 certified production process
  • qPCR & digital PCR assay development, validation, manufacturing, and testing can be outsourced to Microsynth

 

Excellent Technical Support
  • Trained and experienced scientists are happy to support you
 

Dual-Labeled Probes

TaqMan probe

 

The so-called TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. 

In the hybridized state the proximity of the fluorophore and the quencher is sufficient to quench the fluorescence. During the PCR the 5’-3’ exonuclease activity of the Taq polymerase is used to cleave the dual-labeled probe. Thereby the fluorophore and the quencher are separated and a fluorescence signal permits quantitative measurement.

The TaqMan probe significantly increases the specificity of detection. 

We would be happy to assist you with the design of the probes and the associated primers.

 
 
Minimum Yields for Dual-Labeled Probes from Microsynth
5‘ Label 3‘ Label 0.04 µmol scale1,2 0.2 µmol scale1,2 1.0 µmol scale1,2,3
FAM
HEX
YYE
TET

TAMRA
BHQ-1
Dabcyl

1 OD
5 nmol
(FAM only)

2 OD
10 nmol
12 OD
60 nmol
AlexaFluor 350
JOE
VIC
ATTO 425
ATTO 488
ATTO 532
BHQ-1 n/a 1.5 OD
7.5 nmol
8 OD
40 nmol

ATTO 647N
ATTO 620
ATTO 550
(equivalent to NED)
Cy3/Cy5
Dyomics 681
ROX
TAMRA
Texas Red
CalRed
(equivalent to Texas Red)

BHQ-2 n/a 1.5 OD
7.5 nmol
8 OD
40 nmol
1 The synthesis scale represents the initial amount of 3' bases (starting material).
2 Yields indicated in OD260 apply to a probe consisting of 20 DNA bases; Calculation: 1 OD = 5 nmol (please note that this calculation is based on sequences with virtually homogeneous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.)
3 For PAGE purified Probes in the 1.0 µmol synthesis scale, guaranteed yield is 5 OD

 

Potential Applications

  • Demanding qPCR applications with a need for greater flexibility in sequence design, e.g. for targets that are AT-rich and require longer probes (≥25 bases)
  • Digital PCR analysis where the double-quenched probe facilitates discrimination of positive vs negative droplets (improved cluster)

 

Double-Quenched Probes

 

The usual qPCR probes for 5'-nuclease assays have a fluorophore at the 5 'end and a suitable quencher at the 3' end which, on the one hand, clears the fluorescence of the dye and, on the other hand, prevents the prolongation of the probe during the qPCR reaction. For longer probes and thus a greater distance between fluorophore and quencher, the background often increases to an undesirable degree.

Double-quenched probes reduce the background and increase the sensitivity. By incorporating an additional quencher into the sequence near the 5'-fluorophore, a significant improvement in the results can be achieved.
The new quencher IQ-500 with an absorption maximum of approx. 500 nm and a quenching range of approx. 450 nm to 550 nm is designed as a 3 'and internal modification.

Double-quenched probes contain a 5'-FAM fluorophore, a 3'-BHQ-1 quencher and an internal IQ-500 quencher at a distance of 8-10 bases from the fluorophore.

We would be happy to assist you with the design of the probes and the associated primers.

 
Minimum Yields for Double-Quenched Probes from Microsynth
5‘ Label 3‘ Label 0.04 µmol scale1,2 0.2 µmol scale1,2 1.0 µmol scale1,2
FAM IQ-500 with BHQ-1 or TAMRA n/a 2 OD
10 nmol
8 OD
40 nmol

1 The synthesis scale represents the initial amount of 3' bases (starting material).

2 Yields indicated in OD260 apply to a probe consisting of 20 DNA bases; Calculation: 1 OD = 5 nmol (please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.)

 

Potential Applications

  • Demanding qPCR applications with a need for greater flexibility in sequence design, e.g. for targets that are AT-rich and require longer probes (≥25 bases)
  • ddPCR analysis where the double-quenched probe facilitates discrimination of positive vs negative droplets (improved cluster)

LNA Probes


Locked Nucleic Acid (LNA) has been proven to be a powerful tool in many qPCR and digital PCR applications.  When incorporated into DNA oligonucleotides, the resulting LNA containing oligonucleotides offer following main benefits compared to native-state DNA bases only:

  • Increased thermal stability and hybridization specificity
  • More accurate gene quantification and allelic discrimination
  • Easier and more flexible designs for problematic target sequences

Each LNA base addition in an oligo increases the Tm by approximately 2-4 °C and locked nucleic acid probes can be designed to have a ΔTm of >15°C. Usually not more than 5 LNA bases are incorporated into a probe sequence. When designing and optimizing your LNA probes, please follow the guidelines described here.

At Microsynth you can order your LNA probe with the full range of dyes and quenchers we are offering as well for our dual labeled probes.

 

Molecular Beacons

 
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure. The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. Therefore, a fluorophore at the 5' end and a quencher at the 3' end are in immediate proximity and do not fluoresce. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly.

MGB Probes

 
Microsynth now offers customized high quality MGB probes for R&D purposes at affordable prices.
 
DNA probes conjugated with minor groove binder (MGB) form exceptionally stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization-based assays.
 
Therefore, an MGB probe offers following advantages over conventional single or double-quenched probes: 
- higher target binding selectivity
- less background fluorescence
- higher quality of the probe due to shorter length
 
Minimum Yields for MGB Probes from Microsynth
5‘ Label 3‘ Label 0.04 µmol scale1,2,3 0.2 µmol scale1,2,3 1.0 µmol scale1,2,3
FAM
JOE
Yakima Yellow
HEX
MGB-Q5304

1 OD
5 nmol
2 OD
10 nmol
10 OD
50 nmol
 
1 The synthesis scale represents the initial amount of 3' bases (starting material).
2 Yields indicated in OD260 apply to a probe consisting of 20 DNA bases; Calculation: 1 OD = 5 nmol (please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.)
3 MGB Probes ordered as PAGE might receive significantly lower yields.
4 In addition, Microsynth offers the cost-effective 3’ MGB-Q500 quencher moiety. This quencher is only available with FAM as reporter dye and may represent a good entry. For higher demands (top quenching, multiplexing) 3’ MGB-Q530 is the quencher of choice.

 

Design Service

 
Low complexity designs

Dual-labeled probe assays are the most common in the field of real-time PCR. Dual-labeled probe assays are usually more specific than SYBR Green assays. Setting up a probe-based assay also requires fewer optimization steps and the analysis results are easier to interpret. By using well-designed, high-quality probes in your analysis, you reduce the cost of assay optimization and possible error correction and repetition of your measurements compared to SYBRGreen assays, making your analysis faster and more cost-effective.

Due to the availability of various free online software tools, good assay design for a singleplex assay involving a dual-labeled probe (also called hydrolysis or TaqMan probe) is easy to perform and does not require detailed knowledge (it is sufficient to keep in mind following key design guidelines).

Microsynth can recommend the following design tools that make it easy to design highly customized primers and dual-labeled probes for routine qPCR applications:

  • Primer-BLAST

    Primer-BLAST was developed at NCBI to help users make primers that are specific to intended PCR target. It uses Primer3 to design PCR primers and then uses BLAST and global alignment algorithm to screen primers against user-selected database in order to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause non-specific amplifications. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13:134.

  • Primer3Plus

    Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386

 

More complex designs (on request)

When planning experiments requiring more complex assay design considerations, such as splice-specific, multiplexing, SNPs, and CNV designs, or special chemistry (LNA/MGB/Propynyl) you can rely on Microsynth’s experience in this field (>2 decades) as well as selected professional design tools for such demanding applications.
If you need additional assistance with designing primers and probes, contact us at myproject@microsynth.ch. Please do not forget to specify following items:

  • Experimental goal (expression analysis, detection of pathogen, etc.)
  • Accession number(s) or alternatively the gene sequences of your target
  • Desired synthesis scale as well as 5’- and 3’-labels

 

 

How to Order

 

  • Enter our webshop
  • Click on DNA in the "DNA/RNA Synthesis" domain
  • Select either Normal Entry in order to type or copy/paste the desired sequence information etc. or alternatively select Upload Entry by using our convenient Excel template (can be downloaded during ordering)
  • Single-quenched probes (TaqMan, Molecular Beacon, MGB and LNA probes): Select your desired 5', 3' modification as well as HPLC purification1, and your oligo is recognized and processed as a single-quenched probe.
  • In case of LNA probes include the ordering symbols “5, 6, 7, and 8” into your sequence and choose “LNA-A, LNA-C, LNA-G, and LNA-T” under “Inner Modification (5=…)” etc.
  • Double-quenched probes: Include ordering symbol "5" into your sequence (e.g. catattgaa5actgggttaacggaatt) and choose "Internal Quencher 500" under Inner Modification ("5=…"). Select your desired 5', 3' modification as well as HPLC purification, and your oligo is recognized and processed as a double-quenched probe.
  • Follow the further instructions

1 Microsynth delivers qPCR probes of the highest quality. The majority of Microsynth qPCR probes are purified by HPLC whereas a few require PAGE purification in order to meet the stringent QC specifications. If your dye/quencher combination is not compatible with HPLC just use PAGE.