While mass spectrometry (MS) has become ubiquitous in proteomics studies, the use of the technique in other fields such as genotyping has been somewhat limited due to the difficulty of introducing the oligonucleotides into the analyser.
Before a molecule can be analysed by an MS instrument it needs to be ionised and sucked into the gas phase.
Smaller molecules tend to ionise more readily than oligonucleotides and are easier to analyse as they give sharp signals compared to broad signals generated by the large number of isotopes contained in large oligonucleotide.
The new research, published as an early view article in the journal Analytical Chemistry , describes a method that overcomes this problem by combining photo-cleavable mass tags with a simple binding system that enables easy purification of the probe-oligo complex before analysis that promises to speed up and simplify the sample preparation step.
The mass tags act as surrogate markers for the oligonucleotides such that the probes that bind to the single strands of DNA contain a different tag that enables the different genetic variations (single nucleotide polymorphisms or SNPs) to be easily labelled.
Because of the sensitivity of the MS technique, the use of mass tags enables the multiplex detection of labels that are very similar in mass and hence the simultaneous detection of a wide range of SNPs.
In contrast, the fluorescence labels used in PCR and microarray genotyping experiments are rather broad and this limits the number of SNPs that can be looked for at a single location, whether in a microarray or a well-plate used during a PCR experiment.
The mass tag approach uses tagged oligonucleotides as primers for the PCR (polymerase chain reaction) in which different SNP alleles are amplified using primers with tags of different molecular weights.
The latest research was conducted by scientists from the Russian Academy of Sciences in Moscow, the UK's Oxford University and Tridend Technology, a division of the UK's Oxford Gene Technology (OGT), replaces the potentially laborious minicolumn purification and separate cleavage steps "largely reducing sampling handling".
The technology has since been licensed to MassTag Technologies, which was spun out of Oxford Gene Technology at the beginning of the year, and is now in the final stages of organising venture capital (VC) funding.
According to John Thornback, CEO of MassTag Technologies, the company is aiming to have its first products on the market soon.
"In MS you are dealing with very precise molecular masses that enable a very high level of multiplexing, as opposed to fluorescence where you have only a limited bandwidth," said Thornback.
"Also it's a very sensitive detection method that enables multiple applications; one example is that you can use this [mass tag] technology for tissue imaging looking for multiple biomarkers from a sample."
Qiagen Genomics has already commercialised a genotyping method based on using mass tags in its MassCode technology that it claims enables the genotyping of over 40,000 samples per day.
Qiagen claims that its system can genotype more than 40,000 samples per day on a single mass spectrometer.
First the different SNP alleles are amplified using primers tagged with different mass labels before any unreacted mass labels are washed away as unincorporated tags will give the same readings in the MS instrument as the PCR products and could potentially render the experiment meaningless.
In the Qiagen method this is achieved by column chromatography. The tags are then cleaved from the purified PCR products using a mercury UV light before analysis using a quadrupole mass spectrometer which detects the ratios of the different tag species present in the sample to a lower detection limit in the femtomolar range (10-15M).
The Tridend method uses a similar approach except that incorporated into the reverse primer for the 5'-end of the fragments is a disulphide group that enables immobilisation to a gold surface.
This means that any unincorporated tags can be readily washed away from the immobilised double stranded DNA fragments.
The system uses functionalised triphenylmethyl (or 'trityl') mass tags that are readily cleaved from the probes that attach to the 3' end of the oligomers by the MALDI laser ionisation source used at the front end of a MS system.
While the researchers did not quote a lower detection limit for the technique, the ease of cleavage of the labels would suggest it is comparable to other methods.