Licence-free MGB probes for any application

 

Minor groove binder (MGB) probes are dual-labeled 5' hydrolysis probes consisting of a 5' fluorescent reporter dye and a 3' Eclipse Dark Quencher (EDQ) conjugated to a MGB moiety. Biosearch Technologies offers high quality yet cost effective MGB oligos with a competitive turnaround time and no minimum order quantity.

 

illustration of MGB probe

 

 

Short probe design delivers high specificity and signal-to-noise

The 3' MGB moiety binds non-covalently to the minor groove and stabilises the target-probe duplex, effectively increasing the Tm of the duplex. The increased Tm allows probes to be shorter, which increases sequence specificity.

A shorter probe has greater quenching efficiency because the dye is closer to the quencher. Combined with a non-fluorescent quencher, such as EDQ, the short MGB probe leads to low background and high signal-to-noise.

Key benefits

  • Short probes for high specificity: The MGB moiety at the 3' end increases the melting temperature (Tm), allowing a shorter probe and improved sequence specificity.
  • High signal-to-noise for increased sensitivity: The increased quenching efficiency of MGB probes improves sensitivity with lower background noise and a higher signal-to-noise ratio.
  • Improved SNP detection: High duplex stability leads to enhanced ∆Tm and improved SNP/mismatch discrimination.
  • Trusted oligo supplier: Biosearch Technologies is one of the most tenured oligo houses. We are known for quality, consistent delivery and flexibility in format.
  • ISO 9001 and ISO 13485 manufacturing options: Experience freedom from the constraints and restrictions of your current oligo supplier by partnering with us from assay design to scale up and commercialisation.

 

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Improved SNP/mismatch discrimination

Single base mismatches have a greater destabilising effect on MGB probes than other probe types, particularly when the mismatches are within the minor groove binding region. This greatly enhances the ∆Tm for a mismatch and improves SNP discrimination.

 

Trusted supplier of high quality oligos

Our multi-site oligo manufacturing operations provide manufacturing redundancy, risk mitigation and surge capacity. With our unique vertical integration, our chemical manufacturing labs produce most of the critical raw materials used in our own oligo synthesis methods, ensuring a steady and secure supply chain as well as consistent quality at every stage of development. By manufacturing our own dyes, quenchers, specialty amidites, CPG and specialty synthesis columns, we ensure that we are a reliable partner from project inception through commercial scale up.

 

Manufacturing options under ISO 9001 and ISO 13485

We meet your regulatory needs from research and development to scale up and commercialisation with ISO 9001 (RUO grade) and ISO 13485/cGMP (diagnostic grade) certified manufacturing options, as applicable.

 

Dye-quencher options

5' fluorescent dye Abs (nm) Em (nm) 3' quencher
FAM 495 520 EDQ*
TET 521 536 EDQ
CAL Fluor Gold 540 522 544 EDQ
CIV-550 530 550 EDQ
HEX 535 556 EQD
CAL Fluor Orange 560 538 559 EQD

*Eclipse Dark Quencher (EDQ)

 

Equivalent performance to industry standard

MGB probes manufactured at Biosearch Technologies demonstrate equivalent performance to industry-standard MGB probes.

 

MGB assays figure Figure. MGB assays were designed for the E. coli gene lacZ. The probes for each assay were synthesised 5’ FAM, TET, CIV, VIC, HEX or NED and 3’ MGB – Eclipse Dark Quencher (EDQ), by both Biosearch Technologies (blue) and Competitor X (purple). All non-labelled oligonucleotides were synthesised by Biosearch Technologies. The performance of each assay was monitored by running a dilution series (1 x 102 to 1 x 108 copies per reaction) of E. coli genomic DNA in TaqMan™ Fast Advanced Master Mix, following manufacturers recommendations for assay concentrations and cycling conditions. Assays were run on a BIORAD CFX96 (FAM, TET and CIV/VIC) or an ABI QuantStudio5 (HEX/NED). The Cq values for the Biosearch Technologies and Competitor X manufactured assays were compared for each dilution point and shown to have equivalent performance, with PCR efficiency and R2 values passing PCR criteria (efficiency 90-110% and R2 > 0.98) for all dyes.

How to order MGB probes

Select from one of the three formulation options to automatically download the respective excel order form, where further ordering instructions are provided.

We offer MGB probes in solution, dried in individual tubes, or as ValuMix assays. All MGB probes are RP-HPLC purified, and sequence verified with mass spectrometry. Certificates of analysis are available upon request.

Combine convenience and consistency with proven chemistry

MGB ValuMix assays for gene expression analysis and qPCR offer the convenience of having a custom, dual-labeled MGB probe and primer pair delivered in exact amounts within a single tube at your selected probe-to-primer ratio (between 1:1 to 1:4.5). This user-friendly delivery format simplifies your qPCR setup by reducing pipetting steps for rapid, reaction setup times.

MGB ValuMix assays for SNP genotyping include two custom MGB probes and a primer pair delivered at exact quantities within a single tube. Save time and money by reducing pipetting errors and repeat experiments with an optimal blend of high quality primers and probes at an affordable price.

 

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MGB probe technology overview

MGB probes have several advantages over standard DNA probes, making this probe type a powerful, versatile research tool.

Applications of MGB probes include:

  • Investigating gene expression changes in the context of health and disease
  • Detecting pathogens in clinical samples
  • Genotyping to support trait selection to optimise crops, livestock, and aquaculture.
petaluma

 

MGB probes are fitted with an Eclipse Dark Quencher (EDQ) and minor groove binder (MGB) at the 3′ end. The minor groove binder is a dihydropyrroloindole-carboxylate (CDPI3) tripeptide that folds into the minor groove of the target sequence. MGB stabilizes van dar Waals forces, which increases the binding affinity between the probe and target and increases the melting temperature (Tm).

With the added stabilization, the probe itself can be designed significantly shorter. A shorter probe increases quenching efficiency by bringing the fluorophore and quencher closer together, resulting in lower background and higher signal for greater sensitivity and precision. Greater sensitivity results in earlier Cq values, requiring less sample and reagent to achieve the same level of detection.

MGB probes allow for greater assay design flexibility and can support detection of more difficult target sequences such as AT-rich sequences. The probe- sequence is agnostic, which is useful when probe base conversions are limited. Single base mismatches have a greater destabilizing effect on MGB probes, particularly within the minor groove binding region, and enhances ∆Tm for improved SNP detection and allelic discrimination assays.

Using MGB probes enables a shorter probe, starting at only 8 bases in length. Our MGB probes can be extended up to 30 bases in length. If you are designing a probe that is more than 25 bases in length, we recommend using BHQnova probes.

 

Design considerations

qPCR and gene expression

oligo synthesis
  • Target a probe length between 8 and 30 bases.
  • Target a %GC content between 30% and 80%.
  • Avoid runs of identical nucleotides.
  • Do not place a guanosine on the 5′ end. A guanosine next to the reporter dye will alter the fluorescence.
  • Avoid guanosine on the 3′ end (ex. 5′-...GGG-MGB-3′ or 5′-...GGAG-MGB-3′).
  • Avoid 4 consecutive guanosines as these can form a stable secondary structure.
  • For FAM-labeled probes, avoid a guanosine in the second position on the 5′ end.
  • Avoid 6 consecutive adenosines anywhere in the probe.
  • Avoid 2 or more cytidines in the middle of the probe, which can sometimes reduce signal.

Additional considerations for SNP genotyping

  • Melting temperature (Tm) difference between probes should not exceed 1 °C.
  • The SNP site should be located in the middle third of the probe sequence or toward 3′ end, but not in the last two bases of 3′ end.

MGB technical specifications

MGB probe specifications
PROBE LENGTH 8-30 bases
DYES AVAILABLE FAM, TET, CAL Fluor Gold 540, CIV, HEX, CAL Fluor Orange 560
QUENCHER AVAILABLE Eclipse Dark Quencher (EDQ)
YIELD 10, 20, 60 nmols delivered
PURIFICATION RP-HPLC
DELIVERY FORMAT Dry or in solution
QUALITY CONTROL MS and UHPLC
QUALITY STANDARD ISO 9001 or ISO 13485
SHELF LIFE 12 months from date of manufacture
SHIPPING CONDITIONS Ambient temperature or dry ice (in solution)
MGB ValuMix Assay for qPCR and gene expression
PROBE LENGTH 8-30 bases
DYES AVAILABLE FAM, TET, CAL Fluor Gold 540, CIV, HEX, CAL Fluor Orange 560
QUENCHER AVAILABLE Eclipse Dark Quencher (EDQ)
YIELD 0.5 nmol probe/0.5-2.5 nmol primer (FAM)
5 nmol probe/5-22.5 nmol primer
20 nmol probe/20-90 nmol primer
PURIFICATION Probe – RP HPLC
Primers – salt-free
DELIVERY FORMAT Dry
QUALITY CONTROL MS and UHPLC
QUALITY STANDARD ISO 9001
SHELF LIFE 12 months from date of manufacture
SHIPPING CONDITIONS Ambient temperature
MGB ValuMix Assay for SNP genotyping
PROBE LENGTH 8-30 bases
DYES AVAILABLE FAM/TET
FAM/CAL Fluor Gold 540
FAM/CIV
FAM/HEX
FAM/CAL Fluor Orange 560
QUENCHER AVAILABLE Eclipse Dark Quencher (EDQ)
YIELD 2 nmol probe/9 nmol primer
5 nmol probe/22.5 nmol primer
12 nmol probe/54 nmol primer
PURIFICATION Probe – RP HPLC
Primers – RPC
DELIVERY FORMAT Dry
QUALITY CONTROL MS and UHPLC
QUALITY STANDARD ISO 9001
SHELF LIFE 12 months from date of manufacture
SHIPPING CONDITIONS Ambient temperature

Storage conditions

MGB probes are shipped at ambient temperature. Upon receipt, store at +2 to +8 °C. Once rehydrated, the oligonucleotides should be aliquoted and stored at -30 °C to -15 °C. Multiple freeze-thaw cycles (>10 cycles) should be avoided. Probes should be protected from light.

Reaction volumes and number of reactions per vial

MGB probes are available in 10, 20, and 60 nmols delivered. Our Reaction Estimator is a useful tool to calculate the number of reactions that can be performed, depending on desired reaction volume and oligonucleotide concentration. For example, 10 nmol of MGB probe is sufficient for 2,500 x 20 uL reactions containing 200 nM probe.

Oligonucleotide resuspension

Re-suspend the dried probes to make stock solutions. We recommend creating stocks of 100 µM. TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8) is recommended but other molecular biology-grade, nuclease-free diluents may also be used. If another concentration is desired, please see our Oligonucleotide Resuspension Calculator for resuspension assistance.

MGB resources

 

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MGB FAQs

Are minor groove binder (MGB) probes available through LGC Biosearch Technologies?

Yes. Find out more about our MGB probes here

 

What is the minor groove binder (MGB) moiety?

MGB probes are fitted with an Eclipse Dark Quencher (EDQ) and minor groove binder (MGB) at the 3' end. The minor groove binder is a dihydropyrroloindole-carboxylate (CDPI3) tripeptide that folds into the minor groove formed by the probe and target sequence.

 

I am currently using dual-labeled BHQ™ Probes, BHQplus™ Probes, and LNA probes, can I switch to MGB probes?

Switching to MGB probes will require redesign because the MGB moiety increases the melting temperature (Tm) of the probe. Although LNA, BHQplus and MGB probes enhance the stability of the target-probe duplexes, they use different technologies to do so, and this must be considered when designing probe sequences.

 

I am currently using dual-labeled BHQ Probes with the BHQ-1 quencher, can I use the same dyes when I switch to MGB probes?

Yes. The quenching ranges of BHQ-1 and EDQ (the quencher on MGB probes) are similar and therefore the same fluorophores can be used if you switch from dual-labeled BHQ-1 Probes to MGB probes.

 

What purification is recommended for MGB probes?

Our MGB probes are purified by RP-HLPC. MGB probes are not compatible with AX HPLC purification or dual HPLC (which is AX HPLC plus RP HPLC).

 

What is the recommended way to re-suspend and store my MGB probes?

MGB probes are shipped at ambient temperature. Upon receipt, store at +2 to +8 °C. Re-suspend the dried probes to make stock solutions. We recommend re-suspending in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8), but other molecular biology-grade, nuclease-free diluents may also be used. Once rehydrated, the oligonucleotides should be aliquoted and stored at -30 °C to -15 °C. Multiple freeze-thaw cycles (>10 cycles) should be avoided. Probes should be protected from light.

 

What is the difference between dual-labeled BHQ Probes, BHQplus Probes, LNA probes, and MGB probes?

Dual-labeled BHQ Probes, BHQplus Probes, LNA probes and MGB probes are all dual-labeled 5' hydrolysis probes consisting of a 5' fluorescent reporter dye and a 3' quencher, however there are some important differences:

Dual-labeled BHQ Probes, BHQplus Probes, and LNA probes are modified with a non-fluorescent Black Hole Quencher (BHQ-1, BHQ-2 or BHQ-3) at the 3' end.

BHQplus Probes and LNA probes use advanced duplex-stabilising chemistries to allow for the design of shorter oligonucleotides with relatively high melting temperatures. BHQplus Probes and LNA probes are typically 15-25 bases in length, while dual-labeled BHQ Probes are typically 20-30 bases in length.

Minor groove binder (MGB) probes are modified at the 3' end with the Eclipse Dark Quencher (EDQ) and a MGB moiety. The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes.