|High throughput synthesis of long oligonucleotides|
|HEAD||Catherine Gouyette / email@example.com|
|MEMBERS||Catherine Gouyette, Ingénieur II IP / Helynck Olivier, Technicien Supérieur
Inside platform PF7, we synthesize long oligonucleotides for platforms PF2 (DNA microarrays) and shorter ones as primers for PF1 (Genomics) projects, most of the time selected by steering committees and for all the units of the Institut Pasteur which sometimes, prefer to appeal to our ability (specially in quality controls that we make on all our production) instead of buying the oligos from private companies.
The methodology we use, is based on the phosphoramidite chemistry employed by all the automatic synthesizers and this should not change in the coming years as it is very efficient (given its rapidity and the very good yields in coupling reactions).
We are able to make about 570 oligos (70mers), more if they are shorter (primers) a week for microarrays purposes and sequencing. The platform is also making modified oligonucleotides for other purposes (HPLC purified molecular beacons, siRNA, 5’- and/or 3’- modified oligos by fluorophores or amino-groups).
Since its creation, the platform co-operates with many research teams inside this Institute,working on Entamoeba histolytica, Plasmodium falciparum……
or by taking part in international programs on big projects such as: Candida glabrata. Legionnella pneumophila…..
Quality controls are based upon capillary electrophoresis, high performance liquid chromatography, whatever is the length of the oligos and for oligos up to 40-50 bases, a third control is based on mass spectrometry after matrix laser desorption.
We synthesized, (beside standard oligos)
.5’-amino-oligos covalently fixed on an aldehyde modified glass support, to develop microarrays dedicated to the analysis of expression profile of micro RNAs or to create « universal microarrays »
.5’-Cy5 or 5’-Cy3-oligos prepared for making diagnostic microarrays in view of identification of different Plasmodium stems,
.Several milligrams of short oligos for spectroscopic studies: cristallography, X-ray diffraction and NMR (publications with Pr Subirana at the University of Catalunya, Spain) and for the same purpose RNA and DNA oligos for Prof Ghomi (UMR CNRS 7033, Paris 13 University),
.Many molecular beacons, oligos with a 5’-fluorophore group and a quencher at the 3’ end to visualize the mRNA transport in the cell,
.and this year we started synthesis of beacons DNA functionalized by gold nanoparticles to study hybridization with fluorescent DNA targets
Keywords: Oligonucléotides, molecular beacons, siRNA, OMeRNA
C. CACERES, G.WRIGHT, C. GOUYETTE, G. PARKINSON and J. A. SUBIRANA. 2004, A thymine tetrad in d(TGGGGT) quadruplexes stabilized with Tl+/Na+ ions. Nucleic Acids Research, 2004, 32 (3), 1097-1102
J.L. CAMPOS, L. URPI, T.SANMARTIN, C.GOUYETTE, and J.A. SUBIRANA 2005, DNA coiled coils Proc.Natl.Acad.Sci USA,2005,102(10),3663-3666
C. WEBER, G. GUIGON, C. BOUCHIER, L. FRANGEUL, S. MOREIRA, O. SISMEIRO, C. GOUYETTE, D. MIRELMAN, J.Y. COPPÉE, and N. GUILLÉN, 2006. Stress by Heat Shock Induces Massive Down regulation of Genes and Allows Differential Allelic Expression of the Gal/GalNAc Lectin in Entamoeba histolyticaEukaryotic Cell, 2006, 5(5), 871-875
H. BRÜGGEMANN, A.HAGMAN, M.JULES, O.SISMEIRO, M.A. DILLIES, C.GOUYETTE, F.KUNST, M.STEINERT, K.HEUNER, J-Y.COPPÉE and C.BUCHREISER, 2006. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila Cellular Microbiology, 2006, 8(8), 1228-1240
C.PARIS, F.GEINGUENAUD, C.GOUYETTE, J.LIQUIER and J.LACOSTE, 2007. Mechanism of copper mediated triple helix formation at neutral pH in Drosophila satellite repeats
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