James Woodhall, University of Idaho, Parma Research and Extension Center – WPDN
The Communicator: Volume 6, Issue 4, April 2025.
Why consider MGB probes?
MGB probes have a higher melting temperature (Tm) than your typical TaqMan probe such as FAM-BHQ1 or VIC-QSY. If you buy a FAM-BHQ1 probe when the assay requires a FAM-MGB probe your assay will not work as intended. You may still get a signal as the primers will most likely amplify a product, but quantitative accuracy will be significantly compromised as not all target sites are saturated with the probe resulting in reduced fluorescence.
What is an MGB probe?
MGB probes are probes conjugated to a minor groove binder (MGB) group at the 3’ end. The MGB, a dihydropyrroloindole-carboxylate (CDPI3) tripeptide, uniquely folds into the minor groove of the target sequence, enhancing probe stability and performance. Consequently, MGB-conjugated probes have a higher melting temperature (Tm).
Why do they matter?
As MGB probes form extremely stable duplexes, they can allow for shorter probes with high specificity. For example, an MGB probe 12 bp in length can have the same Tm (65°C) as a 27 bp probe (Kutyavin et al., 2000). Short probes also have the advantage of increased quenching efficiency, because the 5’ fluorophore and 3’ quencher are in closer proximity resulting in better signal-to-noise ratio.
Assay design considerations
MGB probes are useful for targeting A/T-rich duplexes and single nucleotide polymorphisms (SNPs) due to the increased stability that the MGB region provides. MGB probes are short, therefore, they can add more specificity because one mismatch in a shorter probe has a larger effect than in a longer probe. MGB probes can be used in multiplexing but no more than two should be included to ensure successful amplification.
Conclusions
If you are buying TaqMan probes it is important to check the protocol to determine whether you need an MGB probe. It can have a massive impact on your assay, especially if you are doing any form of quantification. MGB probes are a useful option for assay design particularly with conserved A/T-rich sequences and for SNP detection.
Literature cited
Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 2000 Jan 15;28(2):655-61. doi: 10.1093/nar/28.2.655. PMID: 10606668; PMCID: PMC102528
