Improved signal-to-noise ratio with EasyBeacons™
EasyBeacons™ and HydrolEasy™ probes are composed of a fluorophore, a quencher and a Hydrophobic Nucleic Acid (HyNA™). HyNa™ is composed of standard DNA nucleotides and Hydrophobic nucleotides.
The Hydrophobic nucleotides of HyNa™ comprise flat hetero aromatic and hydrophobic molecules, which, when they cannot intercalate between base pairs in a complementary duplex, tends to stack on top of each other making hydrophobic interactions.
In this way the system is minimising the hydrophobic surface exposure to water and hence maximising the entropy of the system (this mechanism is known from protein folding, where proteins usually have a hydrophilic outer surface and a more hydrophobic inner core).
When hybridised to a complementary target, the fluorophore and the quencher of EasyBeacons™ and HydrolEasy™ probes are separated in spatial distance allowing the fluorophore to fluoresce.
Hence if there are no or fewer complementary targets than EasyBeacons™ or HydrolEasy™ probes in a mixture, the Hydrophobic nucleotides of the unbound probes will facilitate contact between the two ends of the probe and thereby induce quenching of the fluorophores.
This folding back mechanism is independent of the sequence of the probe, and it is therefore not necessary to design a self complementary stem structure to obtain a proper quenching of the fluorescence in contrast to conventional DNA based molecular beacons.
Example of low background flourescens with EasyBeacons™
EasyBeacons™ and HydrolEasy™ probes have significantly reduced background fluorescence compared to corresponding DNA probes (fluorescence of unbound probe).
In the example shown here two probes, a DNA based probe and a corresponding EasyBeacon™ have been designed to distinguish between a methylated and unmethylated CpG doublet in genomic DNA. The genomic sequence is first treated with sodium bisulphite to convert non-methylated CpG's in the DNA to UpG's while leaving methylated CpG's intact.
The two types of probes have the same maximum fluorescence (at 35ºC),but the background fluorescence of the unbound EasyBeacon™ is significantly lower (approximately 7 times) than the background fluorescence of the corresponding dual-labelled DNA probe.
High Affinity & High Specificity with EasyBeacons™ and HydrolEasy™ probes
Due to the high affinity of HyNa towards complementary DNA, EasyBeacons™ and HydrolEasy™ probes can be made shorter than their DNA counterparts. Generally speaking the shorter a probe is, the better it is to discriminate between single point mismatches.
A typical EasyBeacon™ or HydrolEasy™ probes comprises 1 fluorophore, 1 quencher, between 7 and 18 DNA nucleotides and 2 to 4 IPNs.
The figure below demonstrates that EasyBeacons™ can be designed to achieve a higher specificity than their DNA counterparts. In the example shown here the specificity is 77% higher for the EasyBeacon™ than for the dual labelled DNA probe, measured as the difference in affinity to a matched versus a mismatched target.
Detection of methylation status of single CpG duplets and Endpoint verification with EasyBeacons™
The figures below shows the detection of a single CpG duplet of bisulphite converted genomic DNA and endpoint verification of the amplified product. The results shows a clear separation of the curves of the methylated and unmethylated templates.
Further more, using EasyBeacons™ it is possible to distinguish clearly between a mixture of methylated and unmethylated genomic DNA, pure methylated genomic DNA andpure unmethylated genomic DNA.
Multiplexing and SNP detection with EasyBeacons™
The figures below show multiplex detection of SNP targets using EasyBeacons™. EasyBeacon™ clearly distinguish between SNP and WT target.
