Real-time PCR has rapidly become a standard method for clinical molecular laboratories because of its ability to provide sensitive and highly specific results. Nanogen focuses its research and development efforts on creating innovative technologies and products that provide advanced diagnostic capabilities. To improve PCR performance, Nanogen has developed proprietary minor groove binder (MGB) and Superbase™ technologies for use in real-time PCR primers and probes. All MGB Alert® and Q-PCR Alert™ reagents and kits use Nanogen's MGB approach to real-time PCR, as well as its Superbase technology, to expand assay development capabilities and help overcome previously problematic assay design problems.
Nanogen's MGB approach to real time PCR provides technological advantages over traditional PCR methods including:
Minor Groove Binder (MGB) Technology
Minor groove binders (MGB), a potent class of naturally occurring antibiotics, are long, flat molecules composed of several similar subunits held together by peptide bonds. MGB moieties can adopt a "crescent shape" allowing the moiety to fit snugly into the minor groove, the deep narrow space between the two phosphate-sugar backbones of a double stranded DNA helix. The MGB that Nanogen employs are stabilized in the minor groove by either hydrogen bonds or hydrophobic interactions.
MGB moiety (in red) folded into the minor groove of a double stranded DNA helix.
Nanogen's MGB Alert and Q-PCR probes consist of an MGB moiety attached to the 3'-end or the 5'-end of a DNA probe. The probes leverage the natural ability of MGB moieties to fold back into the minor groove which provides hyper-stability of the hybridization between the DNA probe and target. The MGB moiety acts as a loose fitting lock increasing the temperature needed to melt or disassociate the probe from its target by 15-30℃.
MGB Technology Mechanism
MGB technology uses a hybridization-triggered fluorescent mechanism. When the probe is in solution, the three-dimensional conformation brings the quencher and MGB in close proximity of the fluorescent label, and the fluorescence is quenched. When the probe anneals to a target sequence, the probe unfolds, moving the quencher away from the fluorescent label with the MGB entering the minor grove, allowing a strong fluorescent signal. This means that signal detection occurs during the annealing phase of target amplification. See the illustration below or click here to view an animation on the mechanism of action.
Improved Discrimination
Prior to the introduction of MGB technology, researchers typically needed to increase the size of the probe in order to produce melting temperatures consistent with efficient PCR. Longer probes reduce design flexibility when restricted by small target regions and are less sensitive to mismatch discrimination.
Unlike Taqman probes, Nanogen's patented MGB probes are uniquely designed to overlap with primers for improved results. MGB probes hybridize more strongly to their complementary sequences than regular DNA probes and display increased melting temperature allowing for the use of shorter, yet highly specific, probes.
The use of shorter probes can improve mis-match discrimination as is illustrated below by melting curves of two probe designs (with and without the MGB moiety) specific for the same target. One base pair mis-match has a greater impact on binding for shorter probes than it does for longer probes allowing single base variants to be more easily detected.
Melt Curve Analysis
Nanogen's MGB probes are not degraded during PCR allowing for higher resolution post-PCR melt curve analysis which may be performed in addition to the standard end-point scatter-plot analysis. Melt curve analysis is a valuable tool enabling greater mismatch discrimination and more reliable genotyping. With melt curve analysis, users can confirm target amplification, detect multiple variants presenting different melting temperatures in a single amplification reaction, and detect unknown variants displaying poor amplification plots, reducing missed call and no call results.
Universal Cycling Conditions
Nanogen's MGB Alert probes show an increase in stability of the DNA probe-target hybridization and a subsequent increase in melting temperature (Tm) which, in turn, produces a Tm leveling effect. Regular 8-mer probes containing different G/C and A/T contents display lower Tm as A/T content increases (green bar/line in the graph below). MGB Alert probes stabilize A/T rich duplexes, producing a 'Tm leveling' effect (blue bar/line in the graph below). Hence, the target sequence influence on Tm is reduced allowing a user to run multiple assays under the same cycling conditions.
Advantages of Nanogen's MGB real-time PCR technology:
- Use of short, or highly conserved, specific sequences
- Specific primers and non cleavable probes
- Improved mis-match discrimination with the use of shorter probes
- Post PCR melt curve analysis
- Universal cycling conditions
- Open platform; compatible with most real-time PCR instruments
- Ability to tailor designs to detect most mutations
- Improved primer/probe sensitivity and specificity with the use of Superbases™