Proteus S.a.

Centre National De La Recherche Scientifique

Institut National De La Recherche Agronomique (Inra)

Proteus Gmbh

Jean-Michel Masson

Fabrice Lefevre

Daniel Dupret

Sandrine Dautel

Martine Cerutti

Laurent Fourage

Gerard Devauchelle

Jean François Bloch

Matthieu Chodorge

Cécile Persillon

Jean Fran&ccedil;ois Bloch

Viviane Valverde

Laurence Garnier

Florence Boe

Lyubov Ryabova

The invention concerns a method for detecting a known function, from nucleic acids present in a sample, characterized in that it comprises the following steps: (a) preparing, from the nucleic acids of the sample, nucleic acid molecules comprising the gene(s) coding for the protein(s) corresponding to said function, and the control elements required for the transcription and the translation of said gene(s); (b) in vitro transcription and translation of the nucleic acid molecule prepared in step (a); (c) detecting and/or measuring the function corresponding to the protein(s) produced in step (b).

Method for detecting and/or quantifying a known function from a nucleic acid sample

The invention relates to methods of determining the mutational load of a gene bank obtained by random mutagenesis of a gene of interest by preparing a chart linking the mutational load of a gene bank obtained by random mutagenesis of a model gene with the fraction of mutated model genes observed in the bank; performing, in parallel, random mutagenesis of the model gene used for preparing the chart and the gene of interest to obtain the corresponding mutated gene banks; determining the mutational load of the gene bank obtained using the model gene on the basis of the chart plotted; and applying a correction factor to the mutational load of the mutated model gene bank to determine the mutational load in the gene bank of the mutated genes of interest.

Method of determining the mutational load of a gene library obtained by random mutagenesis of a particular gene and means for implementing same

Ligation-mediated method of recombining polynucleotides in vitro. Polynucleotides from a library are fragmented and the fragments are hybridized to an assembly template. The hybridized fragments are iteratively re-hybridized and ligated until the ends of the hybridized fragments are adjacent to the ends of other hybridized fragments on the assembly template. A final ligation produces recombined polynucleotides.

Process for obtaining recombined nucleotide sequences in vitro, libraries of sequences and sequences thus obtained

The object of this invention is a process for the creation of at least one recombinant polynucleotide sequence comprising a step of oriented ligation of fragments derived from a bank of at least two polynucleotide sequences, and optionally cloning the recombinant polynucleotide sequences, and the selection of polynucleotide sequences offering advantageous characteristics compared to one or several reference sequences.

Process for in vitro creation of recombinant polynucleotide sequences by oriented ligation

The present invention relates to a method of detection in vitro of a target substance in a sample. The sample can comprise, among other things, a nucleic sequence or more generally any type of substance. In particular, the present invention provides a method of detection comprising (i) specificly labeling a substance with a reporter gene and any sequences necessary for the in vitro expression of the reporter gene; (ii) in vitro transcription and translation of the reporter gene; and (iii) in vitro detection of a reporter protein coded by the reporter gene.

METHOD OF DETECTION IN VITRO OF A TARGET SUBSTANCE IN A SAMPLE COMPRISING THE LABELLING OF SAID SUBSTANCE WITH A REPORTER GENE AND WITH THE SEQUENCES NECESSARY FOR THE EXPRESSION OF SAID REPORTER GENE IN VITRO

The invention concerns a method for determining the activity of a substance using a functional test, characterized in that it consists in detecting and/or measuring the variation of a known function corresponding to one or several proteins produced in vitro in the presence and in the absence of said substances or to the substance in the presence or in the absence of one or several proteins produces in vitro.

Process for determination of the activity of a substance using an in vitro functional test

The present invention is directed to a process for the separation and characterization of the functions potentially present in a biological sample containing nucleic acids, characterized in that it comprises the steps of preparation of nucleic acid fragments starting from said sample, association of each one of said fragments with a vector molecule, isolation of each fragment associated with a vector molecule or with a part of each construction composed of a fragment associated with a vector molecule, in vitro treatment of each fragment associated with a vector molecule or of a part of each construction composed of a fragment associated with a vector molecule to obtain transcripts, and testing of the function of the transcripts or of the proteins encoded thereby after translation of said transcripts.

Method for separating and characterizing functions potentially present in a biological sample containing nucleic acids

This procedure comprises the following steps: (1) cleavage of a circular cloning vector comprising two restriction sites a and b giving cohesive ends which are compatible with one another, and two restriction sites c and d which are close to the restriction sites a and b, by restriction enzyme(s), (2) introduction by ligation of the nucleic acid sequence to be polymerized into the linearized vector obtained in (1), between the two restriction sites a and b (1x vector), (3) cleavage of the recircularized vector obtained in (2), by restriction enzyme(s), (4) introduction by ligation of a gene coding for a suppressor tRNA into the linearized vector obtained in (3), between the two restriction sites c and d (1x+s vector), (5) cleavage of the vector obtained in (4) by restriction enzyme(s), (6) introduction by ligation of the fragment a-d obtained in (5) (1x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector at the said sites b and d, (7) introduction by ligation of the fragment obtained in (6) (2x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector, the cleavage being as defined in (6), (8) repetition of step (7), by insertion of the nx+s fragment into a 1x vector, until a sequence containing n+1 fragments is obtained, (9) transformation of a bacterial strain with the vector obtained in (8), and selection of mut+ strains, and (10) extraction of the (n+1)x polymer from the said selected strains.

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Toulouse, France

Jean-Michel Masson