Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-28
2003-02-04
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S068100, C435S069100, C435S091200, C424S093210, C514S04400A, C530S350000, C536S023100
Reexamination Certificate
active
06514703
ABSTRACT:
The present invention has for its object a process permitting separation of the functions potentially present in a biological sample containing nucleic acids and the characterization of said function, by protein expression in vitro after transcription and translation of DNA fragments. The characterization of a function according to the process of the invention can be done among other things by the biochemical analysis of this function and optionally by the cloning or the sequencing of the polynucleotide sequence corresponding to each of the functions possibly present in the studied sample.
In this way, the process according to the present invention more particularly permits the identification and the isolation of genes or of the corresponding proteins possessing a determined or determinable function.
The search for new genes and/or proteins encoded thereby constitutes an important objective of numerous molecular biology laboratories. In effect, the genetic and cellular therapy or the creation of transgenic animals or plants has caused new hopes for human or animal health, diagnoses and feeding, but requires, among other things, the identification of numerous genes and/or protein activities. Thus, numerous techniques exist for the isolation and the screening of genes by cloning and expression.
One of these techniques called “genomics” comprises the sequencing of a part or the totality of the organism's genome, then searching for homology with already identified sequences in data libraries of potentially coding sequences. Once identified, these sequences are to be subcloned and expressed in order to verify that they effectively code for the sought-after property. In addition to the time necessary for its implementation, this technique presents the disadvantage of being capable of identifying only functions homologous to those that are already known and referenced in the databases.
Proteomics is a second possible approach for looking for interesting properties. It consists of extracting the proteins expressed by a microorganism and then purifying them. Each purification fraction is then tested by detecting which one contains the sought-after property. The primary disadvantage of this technique resides in the fact that it does not permit having a direct link between the detected property and the gene that permits the expression of this property. The second disadvantage of this approach is that it does not permit the detection of the properties that have not been induced in the starting microorganism.
A third technique for detecting a function expressed by a microorganism consists of using expression cloning. This method has as a principle the extraction of the DNA of the starting microorganism, fragmenting it and inserting it in an expression vector in vivo that is transformed in a host. This host is selected for its ability to express the genes of the starting microorganism. In the same fashion the vector is always selected for its compatibility with the expression host. The expression cloning technique is used in context of the search for a function in a microorganism having genetic characteristics close to one of the existing host (same codon usage, same GC percentage). In the case of the search for a function starting with a large number of microorganisms of genetically varied origin, the expression cloning method becomes non-usable, the hosts no longer being capable of expressing the heterologous genes that are provided to them.
Additionally, expression cloning like other prior art processes for isolating and screening of genes presents a number of disadvantages:
the cellular toxicity of the transcription and translation products, which can induce genetic recombinations in order to mitigate these toxicity problems,
the poor representativity of the library used,
the implementation time,
the low compatibility between the sequence of the cloned gene and the punctuation of the expression of the vector used,
highly variable expression levels,
codon usage problems,
refolding and post-translational problems,
the problem posed by the influence of the physiological state of a cell on the expression level of the proteins when the protein activity is sought directly in the original cellular extracts,
a high difficulty of automation.
The present invention precisely aims to offset these disadvantages by offering a fast and effective method for the identification of a function associated with a polynucleotide sequence contained in a biological sample containing nucleic acids, and permitting the isolation of said sequence.
This goal is achieved thanks to a process for the separation and the characterization of the functions potentially present in a biological sample containing nucleic acids, characterized in that it includes the following steps:
a) the preparation of nucleic acid fragments starting from said sample,
b) the association of each of said fragments with a vector molecule,
c) the 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 of step (b),
d) the 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 from step (c) in order to obtain transcripts,
e) the test of a function of the transcripts obtained at step (d) or of proteins for which they code after translation of said transcripts.
The process of the invention offers the advantage of enabling the carrying out of:
a test of the properties of the transcripts of step (d), when they have advantageous properties of the tRNA, rybozyme type, or
a test of the properties of the proteins coded for by said transcripts.
In each case where the functions of the proteins encoded by the transcripts obtained at step (d) are tested, the process of the invention comprises at step (d) the treatment of said transcripts in vitro with a cellular extract permitting their translation to protein, then the test of a function of said proteins by any appropriate means.
By function in the sense of the present invention is understood more particularly the property that can be encoded by a polynucleotide sequence. This property can for example be an enzymatic activity or an affinity if said sequences codes for a protein, or an endonuclease activity for example if said sequences codes for a catalytic mRNA.
Thus, the process of the invention permits not only the detection of a function, but also the characterization of this function from a biochemical point of view. If the function is an enzymatic activity for example, it can relate to the analysis of the optimal conditions of functioning (pH, temperature, salts concentration), of the kinetic parameters (Vm, Km), of the inhibition parameters (Ki). If the function is an affinity, it can relate to the determination of the Kd, or of the molecule having the most affinity for this protein. It can also relate to the determination of the size of the translated protein or of the mRNA transcript, and optionally of the sequencing of the corresponding gene.
By biological sample is understood any sample liable to contain nucleic acids, such as a soil, plant, blood, human or animal, water, microbial, cellular or viral culture, biopsy, etc. sample, but these samples can equally correspond to amplification products (PCR, NASBA, etc . . . ), genomic DNA, synthetic DNA, mRNA, or any product of nucleic acids resulting from treatments currently used by a person skilled in the art.
By vector molecule is understood one or several polynucleotide sequences comprising at least a transcription promoter for step (d) and possibly a substance facilitating the isolation of the step (c) fragment. Optionally this substance can be one or several molecules of streptavidine or of biotin, of the polypyrol group, of antibodies, a single or double-stranded polynucleotide sequence, a DNA plasmid vector preferably not containing sequences permitting the in vivo expression of the associated fragment, or any other compound permitting the
Dupret Daniel
Lefevre Fabrice
Masson Jean-Michel
Chakrabarti Arun K.
Fredman Jeffrey
Proteus S.A.
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