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Custom qPCR Probes

 
 

Increase the sensitivity and specificity of your real-time PCR with probes from Microsynth. High-quality qPCR 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 working days on average
 

User-friendly Online Ordering System

  • New and 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
  • Dual-labeled probes with a broad range of dyes and quenchers
  • Double-quenched probes for reduced background and increased end-point fluorescence
  • Molecular beacons and FRET probes
  • Tm enhancing bases as alternatives for MGB and LNA
  • Free design service
  • Assay validation via Microsynth’s qPCR department possible

 

Excellent Technical Support
  • Trained scientists are happy to support you from 8 to 5 pm
 

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 and average yields for dual-labeled probes from Microsynth
5‘ Label 3‘ Label 0.04 µmol scale1 0.2 µmol scale1 1.0 µmol scale1
Minimum yield2 Average yield2 Minimum yield2 Average yield2 Minimum yield2 Average yield2
FAM TAMRA 1 OD
5 nmol
3 OD
15 nmol
2 OD
10 nmol
8 OD
40 nmol
10 OD
50 nmol
30 OD
150 nmol
BHQ-1 9 OD
45 nmol
8 OD
40 nmol
25 OD
125 nmol
HEX
TET
TAMRA n/a n/a 2 OD
10 nmol
8 OD
40 nmol
10 OD
50 nmol
30 OD
150 nmol
AlexaFluor 350
FAM
HEX
TET
Dabcyl n/a n/a 2 OD
10 nmol
5 OD
25 nmol
10 OD
50 nmol
20 OD
100 nmol
AlexaFluor 350
HEX
JOE
TET
ATTO 425/532
Yakima Yellow
(equivalent to VIC)
BHQ-1 n/a n/a 1.5 OD
7.5 nmol
6 OD
30 nmol
8 OD
40 nmol
25 OD
125 nmol
ATTO 647N
ATTO 620
ATTO 550
(equivalent to NED)
Cy3/Cy5
Dyomics 681
ROX
TAMRA
Texas Red
BHQ-2 n/a n/a 1.5 OD
7.5 nmol
4 OD
20 nmol
8 OD
40 nmol
15 OD
75 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.)

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 and average yields for double-quenched probes from Microsynth
5‘ Label 3‘ Label 0.04 µmol scale1 0.2 µmol scale1 1.0 µmol scale1
Minimum yield2 Average yield2 Minimum yield2 Average yield2 Minimum yield2 Average yield2
FAM IQ-500 with BHQ-1 or TAMRA n/a n/a 2 OD
10 nmol
8 OD
40 nmol
8 OD
40 nmol
25 OD
125 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.)

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 to 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.

Design Service

 
Rely on our vast experience in the area of oligonucleotide synthesis and real-time PCR (allele-specific and exon-exon junction overlapping real-time PCR, multiplex kit development, SNP detection) and and let us design and produce your primer probe sets.

How to Proceed:
  • Contact us by e-mail and
  • Provide us with the accession number or alternatively with the gene sequence of your target gene
  • Do not forget to mention the desired synthesis scale as well as 5’- and 3’-labels

 

Conditions:

  • Design service is free of charge for up to 5 primer probe sets per day and customer
If you want to design your primer probes sets on your own, you will find some useful information under Design of Primer Probe Sets in the submenu “Hints and Tips”.

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: Select your desired 5', 3' modification as well as HPLC purification1, and your oligo is recognized and processed as a single-quenched probe.
    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.