Our scientists are pioneers of the bisulphite method used for distinguishing methylated cytosine (Methyl C) residues from unmethylated cytosine (C) residues in DNA. The technology not only serves to identify methylation patterns in the DNA, it can also be used to simplify DNA sequences more generally, particularly in lower organisms. In unmethylated DNA, the bisulphite method converts all cytosines (C) ultimately into thymine (T), so that Cs disappear from the sequence altogether, resulting in the conversion of a 4 base pair sequence into a 3 base pair sequence of only As, Ts and Gs. We refer to this resultant DNA or RNA as ‘3base™’.
The benefits of 3base™ technology can be summarised as:
- Allows for simple multiplexed assays
More targets can be detected per assay
- Universally applicable to all specimen types, compatible with both DNA and RNA
Able to work with multiple specimen types simultaneously
- Compatible with all nucleic acid detection technology
Including PCR, real-time PCR, Isothermal amplification, array based detection, melt curve analysis etc.
- Open platform to suit all laboratories
No need for laboratories to purchase new equipment as 3base™ does not require specialist hardware and is compatible with most automated sample preparation systems and real-time PCR instruments
The conversion is incorporated into standard sample processing protocols and therefore does not require any additional time or treatment steps as compared to standard methods
By eliminating one-in-four of the naturally occurring bases, 3base™ technology is unique in its ability to simplify the detection of an entire class of microbial species. Only one assay is required for all/any variants within a class, thereby providing a new generation screening methodology
- Sodium bisulphite reacts with cytosine to form uracil
- After amplification all uracil residues are converted to thymine
- Previous bisulphite methods resulted in the degradation of up to 96% of starting DNA , whereas the MethylEasy™ technology results in virtually no loss in the starting nucleic acids
- With improved and patented methods, cell lysis and bisulphite treatment of the nucleic acids can be completed in around 15 minutes
Figure 1a. Example of the 3base™ mechanism. The example sequences below show the increase in homology from 75% (“Before”) to 95% (“After”) via the 3base™ conversion where all C bases are detected as T bases.
- Significantly improves consensus primer homology across species variants
- Enables the use of single or minimal primers/probes to accurately screen for all/any variants present in a sample
Figure 1b. The regular and 3base™ DNA sequence for two primers and two probes is shown. The primers and probes for the 3base™ have a more similar melting temperature (Tm) improving the efficiency of multiplex real-time PCR.
- The 3base™ converted genome is different from 4-base native genomes; however genomic aspects that identify the presence of disease or microorganisms remain intact
- The converted genome delivers an increase in target homology (similarity) across sub-species
- Genotyping can be effectively carried out using 3base™ genomes
- 3base™ assays will not cross-react with 4-Base native sequences and this reduces the potential of false positive signals due to impurities in commercially available mastermixes or the environment