We answer your most common questions about MGB Probes

Need high specificity and shorter probes for your hybridisation assays? If you are considering Minor Groove Binder (MGB) Probes for your applications, here are some frequently asked questions to help you understand the benefits and uses of MGB Probes.

What is an MGB Probe?

An MGB Probe is a type of nucleotide probe that includes a minor groove binder (MGB) group. The MGB, a dihydropyrroloindole-carboxylate (CDPI3) tripeptide, uniquely folds into the minor groove of the target sequence, enhancing probe stability and performance.

Figure 1. MGB probes are dual-labelled 5′ hydrolysis probes consisting of a 5′ fluorescent reporter dye and a 3′ Eclipse Dark Quencher (EDQ), conjugated to an MGB moiety.

This group allows the probe to form very stable duplexes with single-stranded DNA targets, making it possible to use shorter probes in hydrolysis probe-based assays.¹ Typically, MGB Probes are 8-30 bases long, with a fluorophore attached to the 5′ end and a quencher attached to the 3′ end. Additionally, the 3′ end is also connected to the MGB moiety that non-covalently binds to the minor groove of double-stranded DNA.

What are the benefits of MGB Probes?

1. Short probes with high specificity

The 3' MGB moiety non-covalently binds to the minor groove of the target, stabilising the probe-target duplex and increasing its melting temperature (T_m). This stability allows for shorter probe designs, which enhances specificity.

2. High signal-to-noise for increased sensitivity¹

The shorter probe design also improves the quenching efficiency, resulting in a low background and a high signal-to-noise ratio.

3. Enhanced SNP detection and allelic discrimination¹

Single base mismatches can have a greater destabilisation effect in MGB Probes compared to other types of probes, especially when these mismatches occur in the minor groove binding region. This significantly increases the change in melting temperature (∆T_m) for mismatches, enhancing the detection of single nucleotide polymorphisms (SNPs).

4. Targeting A/T rich regions

MGB Probes are especially effective at stabilising A/T-rich duplexes compared to G/C-rich ones, which increases the melting temperature (T_m) of the probes and helps simplify their design. According to a study by Kutyavin et al., for a 12mer sequence, attaching the MGB ligand near the A/T-rich 3′-end offered the same stability as adding 15 bases to the 5′-end.¹

Is there anything else that works like MGB Probes?

MGB Probes are dual-labelled hydrolysis probes commonly used in hybridisation assays such as qPCR. While MGB Probes are highly effective, other probe chemistries can also be utilised for similar purposes in real-time PCR applications. These include TaqMan probes, Molecular Beacons, Locked Nucleic Acids (LNAs), and BHQplus™ Probes. Selecting the most suitable probe depends on your specific assay requirements and target sequences. For more information, read our blog – Choosing the best qPCR probe chemistry for your assay.

Are MGB moieties always paired with a quencher?

The MGB and quencher are most often paired together, but the configurations can vary depending on the type of probe and its intended application. In MGB-TaqMan and MGB-Eclipse probes, the fluorophore is at the 5′ end, while the MGB and quencher are attached at the 3′ end.

LGC Biosearch Technologies^TM offers MGB Probes featuring an Eclipse Dark Quencher (EDQ) and an MGB at the 3′ end.

Can the MGB be on either the 3' or 5' end?

Biosearch Technologies offers MGB Probes with an MGB moiety at the 3′ end. Afonina et al. demonstrated that a 5'-CDPI₃ moiety can block primer extension when hybridised to a downstream site, while a 3'-CDPI₃ does not have this effect.²

How much does an MGB increase T_m?

The increase in melting temperature (T_m) due to an MGB depends on various factors, such as the type of MGB, the sequence, and the experimental conditions. When an MGB is incorporated into an oligonucleotide, it stabilises the DNA duplex, leading to a higher T_m.

As an example, an 18 °C increase in T_m was seen for an MGB-15mer oligonucleotide hybridised to a 23mer complement, compared to the unmodified DNA duplex.¹ More extreme increases of up to 49 °C have been observed for A/T-rich octanucleotides.¹

Are there specific types of target sequences/applications where MGB Probes are superior?

MGB Probes are highly valuable due to their higher specificity, shorter length and improved quenching efficiency, thereby providing higher sensitivity. Studies indicate that MGB Probe qPCR can be up to 1,000 times more sensitive than conventional qPCR, significantly enhancing the accuracy of your results.³ These probes are especially useful for detecting short sequences with high specificity. Common applications include:

• SNP genotyping assays

• Allelic discrimination¹

• Detection and quantification of micro-RNA targets⁴

• DNA quantification from crude plant DNA samples⁵

• Pathogenic detection³

Are MGB Probes good for genotyping?

Yes, MGB Probes are an excellent choice for genotyping. Their ability to bind to the minor groove of DNA enhances hybridisation stability, and therefore mismatch discrimination, which is essential for accurate SNP detection. These properties further allow for shorter probe sequences with enhanced signal-to-noise ratio through increased quenching efficiency. Additionally, MGB Probes perform well even with crude DNA extracts, particularly for complex plant genomes, making them suitable for high-throughput applications and cost-effective scaling.

For more detailed information on plant genotyping, you can read How MGB Probes could solve your complex plant genome genotyping issues.

Biosearch Technologies also offer MGB ValuMix assays for SNP genotyping which includes two custom MGB Probes and a primer pair delivered at exact quantities within a single tube.

Are MGB Probes compatible with multiplexing?

Yes, MGB Probes are compatible with multiplex assays. Biosearch Technologies offers MGB Probes featuring a 5' fluorescent dye and a 3' Eclipse Dark Quencher (EDQ) attached to the MGB group. You can customise your dye-quencher combination to meet your specific assay requirements.

No more than two MGB Probes should be included in a multiplex to ensure successful amplification.

For more guidance, you can talk to our experts. Our dedicated technical support team can help you with any questions on our products and to help you integrate them into your workflow (techsupport@LGCGroup.com).

Can MGB Probes be used in combination with other probe types?

Yes, MGB Probes can be used in combination with other probe types. Combining MGB Probes with other types of probes, like BHQ™ (Black Hole Quencher) Probes, has been shown to enhance the specificity and sensitivity of assays, even when working with crude DNA samples. For instance, using MGB Probes in conjunction with BHQ Probe Master Mix has proven effective for SNP detection in plants, providing high specificity and sensitivity without the need for DNA purification.

For more information download our poster on MGB Probes and BHQ Probe Master Mix assay platform.

I’ve seen an MGB Probe in a published paper. What happens if I order this probe without the MGB?

The MGB moiety increases the T_m of the probe, so the probe will require a redesign to have similar performance. Other probe types, such as BHQplus or LNA probes, can also increase binding affinity to the target.

Why may I choose to use an LNA Probe over an MGB Probe?

An LNA (Locked Nucleic Acid) Probe can offer greater specificity and better discrimination against non-target sequences than an MGB Probe, due to their high affinity. Although LNA Probes are more complex to design and may require multiple testing iterations to optimise performance and T_m, they are ideal for applications demanding high specificity.

LNA Probes are employed in real-time qPCR, microarray analysis, fluorescent in situ hybridisation (FISH), as well as oligonucleotide therapeutics. They are particularly suitable for micro-RNA analysis, pathogenic identification, SNP detection, taxonomic classification, etc.

Not sure which probe suits your assay? Try our decision tool for guidance.

For more personalised assistance and integration into your workflow, contact our technical support team at techsupport@LGCGroup.com.

What purification is recommended for MGB Probes?

MGB Probes are purified by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) to ensure high purity and performance. It’s important to note that MGB Probes are not compatible with Anion Exchange HPLC (AX HPLC) or dual HPLC (i.e. AX HPLC plus RP HPLC).

What can I do if the dye I need for my MGB Probe is not listed?

We can potentially make an MGB with different dyes outside our initial offering, by request. Please note these MGB Probes have not been internally tested for performance, therefore we highly recommend performing preliminary studies to ensure probes are suitable for your qPCR requirements.

Can I order my MGB Probe dry or in solution?

MGB Probes can be delivered dry or in TrisHCl, T10E0.1, or T10E1. We do not offer MGB Probes in water.

MGB probes from Biosearch Technologies are designed for optimal sensitivity and specificity, ensuring precise and reliable results in your hybridisation-based assays.

Our multi-site oligo manufacturing ensures consistent quality and a secure supply chain, with ISO 9001 (RUO grade) and ISO 13485/ cGMP (diagnostic grade) certified options for scale up and commercialisation. Customise your probes with a range of fluorophore combinations to meet your specific needs. Visit our website to order now.

For personalised assistance and integration into your workflow, contact our technical support team at techsupport@lgcgroup.com.

References

1. Kutyavin IV, Afonina IA, Mills A, et al. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 2000;28(2):655-661. doi:10.1093/nar/28.2.655

2. Afonina I, Kutyavin I, Lukhtanov E, Meyer RB, Gamper H. Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3199-204. doi: 10.1073/pnas.93.8.3199. PMID: 8622913; PMCID: PMC39582.

3. Teng Q, Shen W, Yan D, et al. Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China. J Virol Methods. 2015;217:64-69. doi:10.1016/j.jviromet.2015.02.025

4. Zhang HH, Wang XJ, Li GX, Yang E, Yang NM. Detection of let-7a microRNA by real-time PCR in gastric carcinoma. World J Gastroenterol. 2007;13(20):2883-2888. doi:10.3748/wjg.v13.i20.2883

5. Ambra R, Marcelli M, D'Orso F. Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe. Genes (Basel). 2023;14(9):1797. Published 2023 Sep 14. doi:10.3390/genes14091797