Unit: High throughput synthesis of long oligonucleotides
Director: Gouyette Catherine
In our laboratory, we synthesize long oligonucleotides, most of the time for DNA microarrays in platform 2 (J.Y.Coppée).Those microarrays are for transcriptome projets approved by the genopole comity :Entamoeba histolytica, Bacillus anthracis, Plasmodium falciparum, Legionella pneumophila, Aspergillus fumigatus, Candida glabrata
In 2003 we made more than 3000 oligos around 70bases long
We made also some short HPLC purified oligos (mg) for cristallography and spectroscopy studies and some siRNA for a study " extinction of multiplication of positive RNA viruses of medical interest (enteroviruses , hepatite C virus) by small interfering RNA "
The platform was created by the end of 2002 : there was an increasing need of long oligonucleotides for DNA microarrays inside the Pasteur Institute and since january 2003 we are located in the Genopole building. We received all our equipment at that moment.
The first part of the year was employed in getting familiar with the different softwares of every apparatus and improving the standard synthesis programs on the Mermade to apply them to long oligos. Mermade IV synthesizers are made for working in 96 wells plate, that is to make simultaneously 96 oligonucleotides..Most of the time we make long oligos and the coupling yield of each base has to be the best possible : for exemple, with an average coupling yield of 99% we have a total yield for a 70 mer of 50%, but with a average coupling yield of 98% (which is not that bad in making short sequences) we get only 25% of the 70 mer( rather than 68% of a 20 mer in the same conditions).The synthesizers need a little bit more than 40 hours to make a 96 plate of 70 mers.
We had also to improve the HPLC gradients and capillar electrophoresis voltage to get faster but significant elugrams for those samples which migrate with more difficulties than shorter oligos in reverse phase or gel capillaries.We use those two techniques to check the quality of the oligos before delivery . Generally on a 96 plate we have now less than 10 oligos which have to be done again :an oligo may seems good in capillar electrophoresis but not in HPLC, separation criteria being different in the two techniques.
Mass spectrometer MALDI-TOF is very reliable with oligos a little bit shorter : it is very useful for HPLC purified oligos made at a larger scale (mg) for cristallography and spectroscopy studies or for RNAi synthesis
Keywords: High throughput oligonucleotide synthesis