Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-15
2003-05-13
Whisenant, Ethan C. (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024310, C536S024330, C536S025300, C536S025320, C435S006120, C435S091100, C435S091200
Reexamination Certificate
active
06562959
ABSTRACT:
The present invention relates to chromosome painting and more particularly the fluorescent probes which can be used in methods such as the FISH (“Fluorescence In Situ Hybridization”) method. The invention also relates to combinations of fluorophores and of optical filters.
In situ hybridization is a technique which makes it possible to detect a DNA (or RNA) sequence by means of a probe having a specific sequence which is homologous to that studied. It is based on the complementarity of the nucleotides (A/T, A/U, G/C) and it can be carried out under precise physicochemical conditions on chromosome or tissue preparations. The result of the in situ hybridization process is the formation of a hybrid between a probe and a target. In situ hybridization includes a denaturing step and also a step for detecting the hybrid or the probe which is carried out after the in situ hybridization of the probe to the target. The sample may adhere in the form of a layer to the surface of the slide and this sample may, for example, comprise or contain individual chromosomes or chromosomal regions which have been treated in order to maintain their morphologies under denaturing conditions. In the context of fluorescence in situ hybridization, the probes are labeled with a fluorophore and the hybridization is revealed by fluorescent labeling.
The recent development of this technique allows the simultaneous visualization, on the same preparation, of several probes each revealed by a different fluorophore. This technique, called multicolor FISH or multi-FISH, has been made possible by the combination of filters specific for the wavelengths of emission of the different fluorescent molecules ensuring the labeling with the aid of a computer-aided imaging carried out by means of infrared-sensitive high-resolution cold CCD cameras (Schröck et al., 1996; Speicher et al., 1996).
The use of probes having a specific sequence homologous to a precise chromosomal sequence or a whole chromosome coupled with the potential for a multicolor fluorescent labeling makes it possible to develop so-called chromosome painting techniques, that is to say to obtain chromosomes of different colors and thus to obtain, if desired, a multicolor complete karyotype. Karyotype is understood to mean the characteristic arrangement of the chromosomes of a cell in the metaphase.
Within the general meaning of the term, “labeling” is understood to mean an entity such as a radioactive isotope or a nonisotopic entity such as enzymes, biotin, avidin, steptavidin, digoxygenin, luminescent agents, dyes, haptens and the like. The luminescent agents, depending on the source of excitation energy, may be classified into radioluminescent, chemoluminescent, bioluminescent and photoluminescent (including fluorescent and phosphorescent) agents. The term “fluorescent” refers in general to the property of a substance (such as a fluorophore) to produce light when it is excited by an energy source such as ultraviolet light for example. “Chromosomal paint probe” is understood to mean a probe or a probe composition such as the probe composition of this invention, which is suitable for hybridizing, under hybridization conditions, with a target which comprises a predetermined chromosome of a multichromosomal genome. If only a fraction of such a chromosome is present in the sample being subjected to such a hybridization with such a probe composition, then this fraction hybridizes and is identified. In practice, a painted probe of this invention may be mixed with a second, a third, and the like, so as to allow the labeling and the simultaneous detection of the two, three, and the like, predetermined chromosomes. The visualization of all the 24 human chromosomes has been made possible by the use of a labeling with a combination of fluorochromes. For example, in the case of the use of 5 different fluorophores, 31 combinations of fluorophores can be obtained. By using this labeling principle and 24 DNA probes specific for each of the human chromosomes, it has been possible to visualize each chromosome differentially. The attribution, by computer processing, of artificial colors to each of the combinations of fluorophores thus makes it possible to color the 24 human chromosomes differently.
Rapidly, the strong potentials of such a multicolor labeling have allowed the analysis of chromosomal aberrations which were difficult to detect up until now by conventional cytogenetic techniques for labeling chromosomes in bands (Summer et al., 1971; Dutrillaux and Lejeune, 1971) (Giema staining, labeling with BrdU, and the like). The principle of labeling of chromosomes in bands is based on the differences in the average base pair composition (richness in GC) between the bands and on the differences in the compaction of the chromatin between the chromosome bands. Chromosome painting has proved to be a very useful tool for detecting interchromosomal aberrations such as translocations, amplified DNA sequences such as the homogeneously stained regions called HSR (HSR for Homogeneously Staining Regions) or the excesses of chromosomal materials such as the marker chromosomes or double-minute chromosomes. Intrachromosomal aberrations such as deletions and duplications will only be detected as a function of the size of the aberrations, if the latter affect the length of the chromosomes, whereas chromosomal inversions will not at all be detectable by this method.
The limits of the use of the current spectral karyotyping as such are due to the fact that it does not make it possible to detect the nature of the chromosome bands involved in an inter- or intrachromosomal rearrangement. To do this, it is essential to couple this technique to the more conventional one of chromosome bands (R or G labeling) such as DAPI counterstaining, Giemsa or propidium iodide staining for example.
The requirement to combine different techniques of course constitutes a handicap in the analysis of chromosomal aberrations and, moreover, the use of the FISH or multi-FISH method which combines the high cost of the apparatus and the instrumentation necessary for the visualization of chromosome painting with the high cost of the probes specific for the chromosomes restricts the possibilities of this technique spreading to research laboratories or to diagnostic laboratories.
Paint probes currently available on the market (GIBCO-BRL, Oncor, Boehringer Manheim and the like) are obtained by DOP-PCR amplification using degenerate PCR primers of chromosomes or for fragments of chromosomes isolated by cumbersome techniques such as chromosome sorting by flow cytometry or microdissection of chromosomes. The hybridization of probes obtained by DOP-PCR does not generate chromosome bands on the chromosomes. The generation of chromosome bands was sought through the creation of artificial bands along chromosomes. This creation of artificial bands requires the use of cumbersome and expensive techniques. Furthermore, it results in bands which are not known reference marks in the field of cytogenetics.
Some others have described the use of chromosome paint probes obtained by amplification of chromosomes by IRS-PCR (Interspersed Repeated Sequences) using primers specific for DNA sequences which are repeated and dispersed in the genome, such as the Alu and LINE sequences. The combined use of LINE and Alu PCR primers for the amplification of human chromosomes by ISR-PCR was previously proposed by Lichter et al., 1990. However, the labeling in R bands which was obtained by the latter does not make it possible to ensure complete painting covering all the regions of the genome, in particular the telomeric regions and certain G chromosome bands.
The object of the present invention is to provide chromosomal probes which can be obtained inexpensively and which, in addition, make it possible to cause quality chromosome bands to appear directly on chromosomes painted in their entirety.
To do this, the present invention relates to probes intended for the labeling of a chromosome, characterized in that they are composed of a set of DNA segment
Genset
Lu Frank
Saliwanchik Lloyd & Saliwanchik
Whisenant Ethan C.
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