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     High throughput synthesis of long oligonucleotides (Platform)


  Director : Gouyette Catherine (oligos@pasteur.fr)


  abstract

 

In our platform, we make exclusively oligonucleotides synthesis but different types of synthesis :

- in 96-well plate format, for DNA microarrays, and in this case oligonucleotides are about 70 bases long

- synthesis made one by one of oligonucleotides of different sizes, modified or not, purified or not by and HPLC, and in amounts going from a few DO units (at 260nm) to some milligrams

- synthesis of purified RNA or siRNA ( checked by HPLC, MALDI-TOF)



  report

cale

a) First of all, a global overlook to the sequences we made in 96-well plates, for programs agreed by the genopole steering commitee  and others :

in the program of developping microarrays devoted to analysis of expression profiles of miRNA (C.Antoniewski), oligos have to be spotted onto another kind of microarrays with a different kind of chemistry and be modified by an aminogroup at the 5' or 3' end of the sequence. We started by making about 40 oligos (50 bases long) 5'NH2 to be chemically bound to the microarray and where the hybridyzation part of the oligo is at the 3' end of the sequence so farther of the microarray. We made too, the same oligos with the hybridyzation part is at the 5' end and the other part at the 3'end with the 3'Amino-group : we had to make very precious HPLC purification to be sure that what'sgoing to be spotted will have the integrity of the hybridyzation part.we made also these oligos 3'OH and 5'OH as negative controls.

As the first results obtained were better with the 3'Amino oligos we made then another whole 96 well plate.

- oligos for the analysis of the virulence of human parasite Entamoeba histolytica with dna microarrays.

- oligos for Plasmodium falciparum, for Plasmodium berghei

- many 96 well plates of primers for the Sequencing Platform PF1 (Maerugin, E.Coli, Leptospires……..)

- a few 96 well plates for Macromolecular Crystallisation and X-ray Diffraction Platform PF6

etc………….

b) As, in the past we were making HPLC purified oligos in the Organic Chemistry Unit, we continue this activity for specific needs of laboratories whom we always worked with as:

- molecular beacons for Pasteur Institute in South Korea and the Cellular Biology of the Nucleus Unit with different quenchers and fluorophores

- oligos with 5'Cy3 or Cy5 fluorophores groups for Pasteur Institute in Cambodia, for the Parasitism Cellular Biology Unit , and Nadine Martinet (CHU Nancy, Cancéropôle Grand Est) to whom me made molecular beacon too

siRNAs for the Enterotropic Viruses and Antiviral Strategies Laboratory.

RNAs for the Drosophile Genetic and Epigenetic G5 and for the Biomolecular and Cellular Physicochemistry Laboratory Paris XIII

DNA oligos for different laboratories in the Institute, or Genescore or Itodys (Jussieu)

and finaly many milligrams of HPLC purified DNA oligos for CRSSA in Grenoble, and for cristallografic studies at the Barcelona University (Pr Subirana)

All these oligos are checked by analytical HPLC and MALDI-TOF spectroscopy

Keywords: oligonucleotides, siRNA, modified DNA sequences, molecular beacon



  web site

puce More informations on our web site


  publications

puce Publications 2005 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
      Helynck Olivier Superior Technician ohelynck@pasteur.fr

Activity Reports 2005 - Institut Pasteur
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