Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-07-24
2001-12-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06333177
ABSTRACT:
FIELD OF THE INVENTION
The current invention relates to a method for selecting oligonucleotides able of identifying differences at the molecular level between a plurality of biological systems. This method has been named “STRIPGEN” for “Selective Technique for Rapid identification of Proteins and GENes”. The STRIPGEN method involves selection of oligonucleotides having a higher affinity and specificity for target molecules of a first biological system and a lower affinity for binding target molecules of a second biological system (and vice versa). STRIPGEN is based on a scheme of positive selection over a first biological system and negative selection over a second biological system. And uses step-wise iterations of binding, separation and amplification. This method is very powerful since it can discriminate very subtle differences between biological systems of the same type such as between normal and abnormal cell types.
DESCRIPTION OF THE PRIOR ART
Many if not all disorders have a genetic background. A genetic disorder finds its origin in the defect of one or a number of genes. The defect is generally a change in the roles of nucleotides in the DNA sequence coding for a protein. Any modification in the normal sequence of a gene (which usually codes for a specific sequence of amino acids in a protein) can after the original sequence of amino acids in a protein. A single defect in a gene can also produce changes in the primary structure of a protein, leading for example to changes in a protein's activity, improper or even absence of protein function, and over or under production of the protein within the cells. As proteins are essential to normal cellular structure and activities, an imbalance in the complex cellular system, even by a single protein, can result in a disease. Genetic defects can be found in either of the 100 000 to 150 000 genes that are distributed across the 23 pairs of human chromosomes. The discovery of causal genes and their protein products is of strategic importance in pharmaceutical terms as they provide powerful means for identifying the most important and significant targets for the development of novel diagnostic and therapeutic products.
Known in the art is a method called SELEX (Systematic Evolution of Ligands by EXponential enrichment). This method, which has been patented in the United States by NeXtar Pharmaceuticals Inc. (Boulder, Colo.) (U.S. Pat. No. 5,712,375), is used for identifying and preparing nucleic acid ligands to tissues. Similarly to the current application, the method described in U.S. Pat. No. 5,712,375 involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification. Although not exemplified, U.S. Pat. No. 5,712,375 also claims for a method wherein a negative selection is performed in order to perfect the discrimination between subtle differences of similar tissue types.
However, unlike the method described in the current application, U.S. Pat. No. 5,712,375 does not suggest a method wherein a step of negative selection is performed Immediately after a step of positive selection. In contradiction to the current method, U.S. Pat. No. 5,712,375 suggests the performance of two or three rounds of negative selections only once a late-round, highly evolved pool of positively selected nucleic acid ligands have been obtained.
Although the method of U.S. Pat. No. 5,712,375 seems powerful, it is limited to biological tissue (single cells or aggregate of cells). Furthermore, since this method lacks an amplification step which the Applicant considers as essential, the method of U.S. Pat. No. 5,712,375 is much less specific and sensitive than the method of the current invention. As a result, the method of U.S. Pat. No. 5,712,375 has been successful only in demonstrating the identification of proteins in a biological tissue (i.e. non-soluble material). Neither this method nor any other method known to the Applicant has successfully shown the identification of soluble and non-soluble proteins which are present in a first biological system but are absent from a second similar biological system.
Therefore, there is a need for a rapid, efficient and simple method for producing oligonucleotides which are highly specific to target molecules, such as proteins, which are present in a first biological system but are absent from a second similar biological system.
SUMMARY OF THE INVENTION
The present invention includes methods for selecting oligonucleotides able of identifying at least one difference at the molecular level between at least two biological systems. These methods are very powerful since they can discriminate very subtle differences between biological systems of the same type such as between cells in different cell cycles, normal and pathological cells infected and non-infected cells, induced and non-Induced cells.
An Important object of the invention is to provide a method comprising the steps of:
a) contacting a pool of oligonucleotides with target molecules of a first biological system, wherein some of the oligonucleotides from the pool have an affinity for at least one of the target molecules of the first biological system;
b) separating the oligonucleotides having an affinity for the at least one target molecule from the remainder of the pool;
c) amplifying the oligonucleotides which have been separated in step b), to yield a pool enriched in oligonucleotides having a higher affinity for the at least one target molecule;
d) contacting the amplified oligonucleotides of step c) with target molecules of a second biological system;
e) removing the oligonucleotides having an affinity for any of the target molecules of said second biological system;
f) amplifying the remaining oligonucleotides to yield a pool of oligonucleotides having a higher affinity for at least one target molecule of the first biological system and a lower affinity for any of the target molecules of the second biological system; and
g) repeating the combination of steps a) to f) until at least one difference is identified.
In a preferred embodiment, the above mentioned method is modified such that, in replacement of step g), the following steps are performed:
g) fractionating the amplified pool of step f) in at least two portions;
h) contacting a first pool's portion with target molecules of the first biological system and separating the at least one target molecule to which oligonucleotides have an affinity from the remainder of the target molecules; and
i) repeating the combination of steps a) to h) until at least one difference is identified, wherein in at least one of the subsequent steps a):
a second pool's portion is used as the pool of oligonucleotides;
only the at least one target molecule separated in step h) is(are) used as target molecule(s) of the first biological system.
Preferably, step h) further comprises the substeps h′) of contacting a third pool's portion with target molecules of the second biological system, and separating any target molecule of the second biological system to which oligonucleotides have an affinity from the remainder of the target molecules of the second biological system; and in step i), for at least one of the subsequent steps d) during the repetition of the combination of steps a) to h), only the target molecule(s) of said second biological system that have been separated in step h′) are used as target molecule(s) of said second biological system.
It is also an object of this invention to provide a method comprising the steps of;
a) contacting a pool of oligonucleotides with target molecules of a first biological system, wherein some of the oligonucleotides from said pool may have an affinity for one or more of the target molecules of said first biological system;
b) removing the oligonucleotides having an affinity for the one or more of the target molecules of the second biological system;
c) amplifying the remaining oligonucleotides, to yield a pool enriched in oligonucleotides having a lower affinity for the target molecules of
Belmaaza Abdellah
Brukner Ivan
Rouissi Noureddine
Horlick Kenneth R.
Polygene Inc.
Strzelecka Teresa
Sughrue & Mion, PLLC
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