Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-06-24
2001-03-06
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091200, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06197501
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to an arrangement of nucleic acid sequences and their use.
With methods of the comparative genomic in situ hybridization (CGH) of reference chromosome preparations of normal Karyotype, it is now possible to determine in a genomic test DNA (for example, tumor DNA, with the suspicion for the existence of unbalanced chromosome aberrations) gains and losses of genoma sections of about 10 Mbp. With amplifications, it is also possible to chart substantially smaller DNA sections by CGH on reference chromosome preparations. These methods are known from Du Manoir, S.; Speicher, M. R.; Joos, S.; Schröck, E.; Popp, S.; Döhner, H.; Kovacs, G.; Robert-Nicoud, M.; Lichier, P.; Cremer, T.; “DETECTION OF COMPLETE AND PARTIAL CHROMOSOME GAINS AND LOSSES BY COMPARATIVE GENOMIC IN SITU HYBRIDIZATION”. Hum. Genet. 90:590-610, 1993 or Joos, S.; Scherthan, H.; Speicher, M. R.; Schlegel, J.; Cremer, T.; Lichter, P.; “DETECTION OF AMPLIFIED GENOMIC SEQUENCES BY REVERSE CHROMOSOME PAINTING USING GENOMIC TUMOR DNA AS PROBE”., Hum. Genet. 90: 584-589, 1993. As genomic reference-DNA, DNA may be used which, if available, can be gathered from cells with a normal chromosome complement thereof or from another person.
With today's state of the art of (CGH, there are two essential limitations. First a further increase of the resolution capability is desirable. It is expected that, with prometa phase chromosomes, CGH analyses of partial trisomy and monosomy with a resolution capability of ≧3 Mbp become possible. This corresponds to an average DNA content of banded chromosomes with a high resolution chromosomal bands with about 1000 bands per haploid chromosome set. However, for many applications, a CGH test would be desirable by which gains and losses of particular genes or even intragenic DNA sections could be safely determined. It is possible that a better resolution can be achieved if the CGH analyses are performed with even more decondensed chromatin structures. On the other hand, CGH for mitotic reference chromosomes have the disadvantage that the fully automatic identification of chromosomes by fluorescence banding for example with DAPI and measurement of the CGH fluorescence quotient is complicated and time-consuming.
It is the object of the invention to provide an arrangement of nucleic acid sequences by which, with relatively little technical expenses automation and substantially improved resolution can be achieved.
SUMMARY OF THE INVENTION
A method of determining the relative number of nucleic acid sequences in test cells which provides for a high resolution during comparative genomic hybridization by keeping all components separate from each other and selecting target nucleic acids which have a high resolution capability for determining genomic imbalances in the test cells and which facilitate a screening of the test cells for over- or under-expression of individual genes.
A substantial improvement with regard to the resolution capability and also with regard to a fully automatic evaluation is achieved by a CNH-matrix test (CNH=comparative nucleic acid hybridization) wherein, in place of mitotic chromosomes, specific nucleic acid sequences (designated below as target nucleic acids, in the case of DNA as target- DNA, in the case of RNA as target RNA) are deposited on a suitable carrier material (designated below as matrix). A target nucleic acid may consist of one or many different DNA- or, respectively, RNA-sequences. The complexity of a target nucleic acid depends on the respective formulation of the question. The CNH-matrix test should facilitate a fully automatic gain or deletion balance of genetic imbalances in a genomic test-DNA wherein the resolution capability for the selected genome sections, for example, individual genes may be in the kbp-range.
The target nucleic acids are immobilized on a solid matrix which consists for example of filter paper or of glass. The area of the matrix in which a target nucleic acid is deposited is designated below as a slot. Subsequently, the simultaneous hybridization of test- and reference-DNA occurs against the target nucleic acids. Alternatively, the hybridization of test- and reference-DNA against the target nucleic acid may also be done in solution. For this, it is necessary to provide a separate hybridization for each target nucleid acid. The evaluation occurs after binding of the hybridization products on a solid matrix or directly in solution.
In contrast to the highly variable arrangement of individual chromosomes in metaphase representations as they are utilized in a comparative genomic in situ hybridization, the position of the genome sections which are to be tested for gains and losses in the test DNA can be clearly determined on a matrix. Furthermore, the sizes and shapes of individual chromosomes differ substantially from metaphase to metaphase, whereas the size and geometry of the particular slots can be standardized. These possibilities of a standardization of position, size and geometry of the target nucleic acid slots facilitate the fully automatic evaluation of a matrix in comparison to CGH of metaphase chromosomes to a great extent. Size and distance of the individual slots can be so selected that the automatic control of a table with the matrix disposed thereon or, alternatively, of a light beam can be easily realized with sufficient precision. If desired, fluorescence quotients within a slot can also be determined in several separate areas and an average can be calculated therefrom.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The invention will be described on the basis of a CNH matrix test for the analysis of imbalances of genomic DNA or, respectively, expressed RNA in various tissues and cell types using seven examples.
For the comparative quantification of the gene expression in various tissues and cell types a test is to be developed which is based on the comparative hybridization of differently marked mRNA or, respectively, cDNA of two tissues or cell types on a matrix with the corresponding cDNA-clones.
The principle of the CNH-matrix test is based on the comparative hybridization of test and reference nucleic acid samples with respect to target samples, which were deposited on glass or on a filter, and the quantitative determination of fluorescence quotients for the hybridized samples. The individual method steps are described below:
1. Selection of test and reference DNA or, respectively, RNA samples.
Genomic test and reference DNA's are selected in accordance with the same criteria as with the CGH tests for metaphase chromosomes. It is possible to use universal genomic DNA or genomic DNA amplified by means of DOP-PCR. This is described for example by Speicher, M. R.; du Manoir, S.; Schröck, E.; Holtgreve-Grez, H.; Schoell, B.; Lengauer, C.; Cremer, T.; Ried, T.: “MOLECULAR CYTOGENETIC ANALYSIS OF FORMALIN-FIXED, PARAFFIN-EMBEDDED SOLID TUMORS BY COMPARATIVE GENOMIC HYBRIDIZATION AFTER UNIVERSAL DNA-AMPLIFICATION”. Hum. Mol. Genet. 2: 1907-1914, 1993. As test and reference samples for comparative tests of the gene expression, mRNA preparations or, respectively, cDNA libraries of selected cells or tissue but also individual cDNA samples and combinations of cDNA samples can be used.
2. Selection of Target DNA or Target RNA
As target nucleic acids, which are applied to the matrix in a way described below, cloned genomic DNA sections of a species (for example, human) can be used, for example DNA preparations of plasmid clones, cosmid clones, Pi-clones, YAC-clones, which comprise genomic sections of a few kbp up to several Mbp. Instead of purified nucleic acid, sorted chromosomes or microorganisms which contain the respective target nucleic acid, can be directly applied to the matrix.
The physical mapping of the samples used should be known. For even larger genome sections such as certain chromosomal bands, mixtures of the DNA of selected genomic DNA clones can be prepared or DNA's of clone combinations can be used which are made from sorted or microdissected ch
Cremer Thomas
Jauch Anna
Lichter Peter
Ried Thomas
Speicher Michael
Deutsches Krebsforschungszentrum
Fredman Jeffrey
Reed Smith LLP
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