Safeguarding sample integrity

New evaporator technology allows oligo drying without degradation

There are many applications in modern molecular biology that require dye labelled oligonucleotides. These include “scorpion” probes1, taqman probes2 and molecular beacons3 all used in SNP genotyping. Since these are predominately used as diagnostic tools, it is of vital importance that they are of high quality. While it is easy to carry out quality control throughout the production process, once the oligonucleotide has been dried ready for shipment the only means of Q.C. is to resuspend the product. This is obviously undesirable. It is therefore important that the drying process is not detrimental to the oligonucleotide.

In this study we demonstrate how the advance of centrifugal evaporator technology has enabled us today to effectively dry dye-labelled oligonucleotides without risking thermal degradation.

The two systems used in this study were a Model SF-60 originally supplied by Genevac 15 years ago and a modern computer-controlled evaporation system, the Genevac HT-4 (Figure 1).

 Figure 1. The GeneVac HT-4

The SF-60 has been in regular use for routine drying at Eurogentec for many years, but is known to be very simplistic and to have several operational drawbacks. Chief amongst these has been the systems inability to control the heat input into the samples, a lack of sample protection from direct heating and inadequate temperature control for temperature sensitive samples. These problems were entirely solved in the sophisticated design of the Genevac Model HT-4, introduced in 2002. For the purposes of Eurogentec, these centrifugal evaporators are used as final drying machines producing a dried product ready for delivery to the customer.

A variety of oligonucleotide probes were selected for the study, each of which were less than 20 bases in length and were modified at the 5’ and/or 3’ end (see table 1). Five of each of the following fluorescently labelled oligonucleotides were used.

Table 1: Fluorescently labelled oligonucleotides used
5’ FAM  5’ Cy5   5’ Cy3  5’ HEX
5’ JOE  5’ TET    5'FAM  3’ TAMRA

Each of the samples were split equally into three aliquots, a control (which underwent no lyophilisation) and the remaining two which were placed in the HT-4 and SF-60 respectively.

The test samples were subsequently evaporated to dryness in their respective instrument, using the standard settings for each run that are in regular use at Eurogentec. In the case of the SF-60, there is very little control of the run, save spin, vacuum on or off and lamps on or off. However, a more controlled and precise evaporation is possible with the HT-4. Using the HT-4, a three-stage run was used for water/acetonitrile samples. In the first stage the vacuum was quickly reduced to 200mbar, then, to avoid bumping, gently ramped from 200mbar to 40mbar at which point the acetonitrile boils off. After this stage, vacuum is reduced to just 8mbar at which point the water is efficiently evaporated without freezing it. When all the water is removed, a final drying stage takes the vacuum down to 2mbar to ensure total dryness in the finished product.
With the HT-4, samples underwent a pre-programmed drying run, while the SF-60 samples were run until the heat lamp switched off, indicating that all the solvent had evaporated, and thus the run was ended manually.

Following the removal of the probes from the instruments, the oligonucleotides were resuspended in 100µl MilliQ water and vortexed until fully dissolved.

Each of the samples were prepared for mass spectrometry analysis using a Dynamo MALDI-TOF instrument powered by a nitrogen laser. The analyte (3µl) was mixed with cation-exchange resin (3µl), and a hydroxypicolinic acid matrix (3µl), which is necessary for the desorption/ionisation reaction. The analyte-matrix mixture (3µl) was then spotted onto a stainless steel target and allowed to crystallise.

The mass spectra for the Control, HT-4 and SF-60 samples were obtained in the positive ion mode, and the acquired molecular weights were compared against the calculated theoretical mass of the test probes to identify whether the sequence and modification were present and correct.

Following the mass spectrometry analysis, the samples underwent analytical HPLC to verify the presence of the dye and its conjugation to the primer, and to illustrate the sample purity. Each sample (20µg) was suspended in a volume of water (120µl) and filtered before being introduced into a Waters ‘Alliance’ HPLC coupled to a photodiode array detector. The analytes were subjected to reverse-phase chromatography, with elution facilitated by 95% acetonitrile/water over a 30 minute gradient.

Results
With the exception of the FAM labelled samples, a significant degradation was observed in all samples dried using the SF-60. By comparison no degradation was observed for samples dried in the HT4. The results of the cy5 labelled oligos are shown in Figure 4. From our experience we believe that the degradation using the SF-60 was caused by a number of factors. These include overheating from direct IR/UV lamp illumination of the tubes containing the samples and a lack of temperature control in the older evaporator design. In the newer HT-4, while the samples are again heated by IR lamp illumination, it is onto the base of the solid aluminium sample holders and not directly onto the tubes. In addition the HT-4 has very sophisticated temperature control, protecting even heat sensitive samples from such overheating.

Conclusions
The results clearly show that the cy5 oligonucleotide was thermally degraded by the drying process used by the older SF-60 system. By comparison the advances in centrifugal evaporator technology encompassed in the Genevac Model HT-4 have enabled Eurogentec to effectivly dry its range of fluorescently labelled oligonucleotides safeguarded from the potential effects of thermal degradation.

By K.S.Trevett and C.M.McKeen , Eurogentec, Belgium
enquiry number 05105

References
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