U.S. patent application number 09/773647 was filed with the patent office on 2001-11-01 for arrangement of nucleic acid sequences and their use.
Invention is credited to Cremer, Thomas, Jauch, Anna, Lichter, Peter, Ried, Thomas, Speicher, Michael.
Application Number | 20010036633 09/773647 |
Document ID | / |
Family ID | 6506399 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036633 |
Kind Code |
A1 |
Cremer, Thomas ; et
al. |
November 1, 2001 |
Arrangement of nucleic acid sequences and their use
Abstract
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.
Inventors: |
Cremer, Thomas; (Heidelberg,
DE) ; Ried, Thomas; (Heidelberg, DE) ;
Speicher, Michael; (Heidelberg, DE) ; Jauch,
Anna; (Heddesheim, DE) ; Lichter, Peter;
(Gaiberg, DE) |
Correspondence
Address: |
Jules E. Goldberg
Reed Smith, LLP
17th Floor
375 Park Avenue
New York
NY
10152
US
|
Family ID: |
6506399 |
Appl. No.: |
09/773647 |
Filed: |
January 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09773647 |
Jan 31, 2001 |
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08669229 |
Jun 24, 1996 |
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6197501 |
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Current U.S.
Class: |
435/6.11 ; 435/5;
435/6.14; 435/91.2; 536/23.1 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6834 20130101; C12Q 1/6827 20130101; C12Q 1/6841
20130101 |
Class at
Publication: |
435/6 ; 435/5;
435/91.2; 536/23.1 |
International
Class: |
C12Q 001/70; C12Q
001/68; C07H 021/02; C07H 021/04; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1993 |
DE |
DE 43 44 726.0 |
Dec 17, 1994 |
EP |
PCT/EP94/04208 |
Claims
What is claimed is:
1. An analytical element for analyzing genomic variations by
comparative genomic hybridization comprising a supporting matrix
having target nucleic acid sequences fixed thereon in a specific
geometric arrangement.
2. An analytical element according to claim 1, wherein the
supporting matrix comprises filter paper, glass or microplates with
preformed cavities.
3. An analytical element according to claim 1, wherein the target
nucleic acid sequences comprise cloned genomic DNA portions of a
species, sorted chromosomes, microdissected chromosome portions,
clone libraries derived from sorted or microdissected chromosomes
of the human species or other species, cDNA probes, or combinations
of mRNA fractions and either cDNA probes or cDNA libraries.
4. An analytical element according to claim 1, wherein the
supporting matrix is a slide and the target nucleic acid sequences
are fixed onto the slide by a thin polyacrylamide film.
5. An analytical element according to claim 1, wherein the target
nucleic acid sequences are fixed onto the supporting matrix by
admixing the target nucleic acid sequences with carrier substances
and subsequently fixing the target nucleic acid sequences onto the
supporting matrix.
6. An analytical element according to claim 1, wherein the specific
geometric arrangement comprises the target nucleic acid sequences
from top to bottom corresponding to the order of their physical
arrangement on a chromosome from p.sup.th to q.sup.th.
7. An analytical element according to claim 1, wherein the specific
geometric arrangement comprises the target nucleic acid sequences
arranged next to each other in parallel rows.
8. An analytical element according to claim 1, wherein the target
nucleic acid sequences comprise nucleic acid sequences representing
the 24 different human chromosomes.
9. An analytical element according to claim 1, wherein the target
nucleic acid sequences comprise human chromosomes 13, 18, 21, X and
Y.
10. An analytical element according to claim 1, wherein the target
nucleic acid sequences represent the human chromosome arms: 1p, 1q,
2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 9q,
10p, 10q, 11p, 11q, 12p, 12q, 13q, 14q, 15q, 16p, 16q, 17p, 17q,
18p, 18q, 19p, 19q, 20p, 20q, 21q, 22q and Yq.
11. An analytical element according to claim 1, wherein the target
nucleic acid sequences comprise bands resulting in a resolution
capability of a cytogenetic banding analysis with 400 or 800
chromosome bands per haploidernic chromosome set.
12. An analytical element according to claim 1, wherein the target
nucleic acid sequences comprise defined subchromosomal nucleic acid
sequences which are specific for gains and/or losses of genomic
sequences characteristic of the cell types being screened.
13. An analytical element according to claim 12, wherein the
defined subchromosomal nucleic acid sequences are selected from the
group consisting of sorted chromosomes, microdissected chromosome
sections, chromosome arms, protooncogenes, tumor suppressor genes
and amplified isolates from cDNA libraries.
14. An analytical element according to claim 13, wherein the
defined subchromosomal nucleic acid sequences comprise genomic
sections of a few kbp up to several Mbp.
15. A method of making an analytical element for analyzing
variations by comparative genomic hybridization comprising
selecting target nucleic acids for hybridization and arranging the
target nucleic acid sequences on a matrix in a specific geometric
arrangement.
16. A method according to claim 15, wherein the target nucleic acid
sequences comprise defined subchromosomal nucleic acid sequences
which are specific for gains and/or losses of genomic sequences
characteristic of the cell types being screened.
17. A method according to claim 16, wherein the defined
subchromosomal nucleic acid sequences are selected from the group
consisting of sorted chromosomes, microdissected chromosome
sections, chromosome arms, protooncogenes, tumor suppressor genes
and amplified isolates from cDNA libraries.
18. A method according to claim 17, wherein the defined
subchromosomal nucleic acid sequences comprise genomic sections of
a few kbp up to several Mbp.
19. In a method for analysis of genomic variances comprising
screening cell types by comparative hybridization using the
analytical element of claim 1.
20. The method of claim 19, wherein the target nucleic acid
sequences comprise defined subchromosomal nucleic acid sequences
which are specific for gains and/or losses of genomic sequences
characteristic of the cell types being screened.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims German Convention priority of DE 43
44 726.0 filed Dec. 27, 1993 and is a continuation application Ser.
No. 08/669,229 filed Jun. 24, 1996, now U.S. Pat. No. 6,197,501
issued Mar. 6, 2001, the complete disclosure of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to an arrangement of nucleic acid
sequences and their use.
[0003] 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.; Schrock, E.; Popp, S.; Dohner, H.;
Kovacs, G.; Robert-Nicoud, M.; Lichter, 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.
[0004] 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 prometaphase
chromosomes, CGH analyses of partial trisomy and monosomy with a
resolution capability of .ltoreq.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.
[0005] 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
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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 an 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
[0010] 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.
[0011] 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.
[0012] 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:
[0013] 1. Selection of test and reference DNA or, respectively, RNA
samples.
[0014] 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.; Schrock, 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.
[0015] 2. Selection of Target DNA or Target RNA
[0016] 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.
[0017] 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 chromosomes of the human or other species.
For comparative tests of the gene expression, cDNA samples,
combinations of cDNA samples or cDNA combinations as well as mRNA
fractions can be used as target nucleic acids.
[0018] Preparation of the CNH Test Matrix
[0019] The target nucleic acids needed for a desired CNH-matrix
test are deposited on a filter in a geometric arrangement as
desired by the tester. The arrangement may for example by such that
the order of genomic target nucleic acids on the matrix from top to
bottom corresponds to the order of the physical arrangement on a
chromosome from pth to qth. Consequently, each sample has a slot
with a well defined position on the filter assigned to it. For
nucleic acid bands, common paper filter can be used. For
fluorescence processes the filters must be so selected that their
properties such as their innate fluorescence will not disturb the
detection of the fluorescence signals. In this way slots for
different chromosomes, chromosome sections and genes can be
arranged side-by-side in parallel columns. The selection of the
target nucleic acids depends on the purpose of the diagnosis and
the desired resolution capability of the CNH matrix tests. A slot
matrix may contain target nucleic acids for expressed sequences or
genomic sections of selected genes as well as target DNA for
chromosome sections, individual chromosomes or even the complete
chromosome set. Their number may vary dependent on the diagnostic
objective from a few to several hundred target nucleic acids. The
target nucleic acids may be single-stranded samples or
double-stranded samples. In the latter case, the target nucleic
acid must be made single-stranded by a suitable denaturization step
before the CNH test is performed. The target nucleic acids must be
bound to the filter by suitable treatment of the filter so that
they remain in place during the CNH procedure.
[0020] For manufacture of the CNH test matrix on glass, a procedure
is required by which the target-DNA or the target-RNA is firmly
bound to the glass. There are already several protocols for this
purpose such as the coating of object carries with a thin
polyacrylamide film and the subsequent immobilization of the
samples to be applied by a process in accordance with Khrapko, et
al. (Khrapko, K. R.; Lysov, Y. P.; Khorlin, A. A.; Ivanov, I. B.;
Yershov, G. M.; Vasilenko, S. K.; Florentiev, V. L.; Mirzabekov,
A.D.: "A METHOD FOR DNA SEQUENCING BY HYBRIDIZATION WITH OLIGO
NUCLEOTIDE MATRIX". DNA-Sequence-J. DNA-Sequencing and Mapping
1:375-388, 1991). Another possibility resides in the admixing of
carrier substances such as proteins which cause only few or
distinguishable background signals to the target nucleic acids, the
application of the mixture to the matrix and a subsequent fixing,
for example by methanol/ice vinegar or formaldehyde. The selection
and arrangement of the samples on the glass matrix are done as
described above. Instead of glass other hard materials could be
used. Microplates with preformed cavities appear to be particularly
suitable.
[0021] In an alternative process for making a CNH-matrix on glass
or on a filter the hybridization is performed in
solution--separately for each target nucleic acid. With this
method, particular attention must be given to the quantitative
separation of the non-hybridized sample molecules. This can be done
by conventional methods such as gel-filtration,
gel-electrophoresis, chromatography or by enzymatic disintegration.
The signal intensities of the test and reference-DNA are measured
only after this separation. This measurement can be performed after
binding of the hybridization products on a solid matrix or in
solution as far as the target nucleic acids are concerned. The
measurement after binding on a solid matrix is performed as
described below; the measurement in solution can be performed
batchwise for each reaction batch or in an automated fashion, for
example in a flow-through spectrophotometer. The signals of the
test and reference nucleic acids can be determined in accordance
with the signal characteristic. For example, test and reference
nucleic acids can be marked by different fluorochromes. In
accordance with the state of the art of fluorometry both can be
excited and measured separately and, in accordance with the state
of the art of the fluocytometry, they can be simultaneously excited
and separately.
[0022] The marking of the nucleic acid samples by haptenes (for
example, biotin or digoxigenin) or directly by fluorochromium is
done by means of molecular-genetic standard procedures (for
example, Nick Translation, Random Priming) as described by Lichter,
P.; Cremer, T.; "CHROMOSOME ANALYSIS BY NON-ISOTOPIC IN SITU
HYBRIDIZATION" in: Human Cytogenetics: "A PRACTICAL APPROACH",
eds.; Rooney, D. E.; Czepulkowski, B. H.; IRL Press, Oxford:
157-192, 1992 and by Raap, A. K., Wiegert, J.; Lichter, P.;
"MULTIPLE FLUORESCENCE IN SITU HYBRIDIZATION FOR MOLECULAR
CYTOGENETICS" in: Technics and Methods in Molecular Biology:
Non-radioactive labeling and detection of bio-molecules; ed:
Kessler, C.; Springer Verlag Berlin, Heidelburg, N.Y.: 343-354,
1992.
[0023] The Comparative Nucleic Acid Hybridization is performed in a
way as described by Du Manoir, S.; Speicher, M. R.; Joos, S.;
Schrock, E.; Popp, S.; Dohner, H.; Kovacs, G.; Robert-Nicoud, M.;
Lichter, Page.; Cremer, T.; "DETECTION OF COMPLETE AND PARTIAL
CHROMOSOME GAINS AND LOSSES BY COMPARATIVE GENOMIC IN SITU
HYBRIDIZATION". Hum. Genet. 90:592-593, 1993 or by Speicher, M. R.;
Du Manoir, S.; Schrock, E.; Holtgreve-Grez, H.; Schoell, B.;
Langauer, C.; Cremer, T.; Ried, T.: "MOLECULAR CYTOGENETIC ANALYSIS
OF FORMALIN-FIXED, PARAFFIN-EMBEDDED SOLID TUMORS BY COMPARATIVE
GENOMIC HYBRIDIZATION AFTER UNIVERSAL DNA AMPLIFICATION". Hum.
Genet. 2: 1913-1914, 1993. The hybridization of RNA samples occurs
in an analog way and under consideration of the precautions common
with RNA hybridizations.
[0024] The hybridized sample sequences are detected by way of
molecules which generate quantitatively determinable signals which
can be sufficiently distinguished from the "background" signals of
the matrix. For this purpose, fluorescent properties are preferred
at this point. With fluorochromium-marked nucleic acids the sample
sequences can be directly detected after the usual washing steps.
Fluorescence detection reactions by haptene-marked nucleic acid
samples is performed in accordance with standard procedures as
described for example by Lichter, P.; Cremer, T. in: "CHROMOSOME
ANALYSIS BY NON-ISOTOPIC IN SITU HYBRIDIZATION" in Human
Cytogenetics: A practical approach; eds.; Rooney D. E.;
Czepulkowski, B. H.; TRL Press, Oxford: 157-192, 1992. Besides
fluorescence other detection methods may be used which will provide
quantifiable signals, such as chemical luminescence,
phosphorescence and radioactivity in order to directly or
indirectly determine the presence of nucleic acids. Different
detection methods for the test and reference nucleic acids may also
be combined in a single experiment.
[0025] Following the CNH procedure, the fluorescence signals are
quantitatively determined for each slot of the matrix (for example,
with a CCD camera) and, from that, the fluorescence quotient test
nucleic acid/reference nucleic acid is calculated by a
microprocessor. The fluorescence quotient is determined as
described by Du Manoir et al. (1993) (pages 592-593) or by Speicher
et al. (1993) pages 1913-1914) with the difference that the
measurements are performed with the aid of masks, not on the
individual chromosomes, but within the individual target nucleic
acid slots. In CNH control experiments with differently marked
genomic DNA from cells with normal Karyotypes or, respectively,
differently marked identical cDNA or RNA samples, the variations of
these quotients which are normally to be expected are determined on
the basis of a predetermined reliability level. With samples having
a genomic duplication or deletion of a chromosome, of a chromosome
section or of a gene which can be determined by the test, a
systematic increase or, respectively, reduction of the quotient in
the slots which contain the respective target nucleic acids is to
be expected. The fluorescence quotient for the remaining slots
however, should remain within the control range.
[0026] Since, in each slot, the hybridization signal resulting from
the test is genome is compared with that resulting from the normal
reference DNA, the CNH matrix test should be relatively insensitive
with regard to variations in the amount of target nucleic acids in
the various slots which occur with the preparation of the matrix.
Variations in the mixing ratio of the tumor DNA and the reference
DNA as they may occur in different experiments have the same effect
on all the quotients and can therefore also be standardized.
[0027] An important aspect is the selection of suitable equipment
for the quantitative determination of the hybridization signals.
The detection instruments should generally be capable, of measuring
linear differences between the signal intensities over a wide
range. For the detection of fluorescence signals various instrument
configurations may be used such as: fluorescence microscopes which
include a (cooled) CCD (Charged Coupled Device) camera or
fluoroscanners, wherein fluorescence scanning is performed by way
of an electronically controlled laser beam and detection occurs by
way of a sensitive photo-multiplier. Also with the flow-through
spectrophotometry excitation is obtained by a lamp or a laser and
detection by way of a photomultiplier. Depending on the type of
detection signals also other methods such as densitometry (see for
example, phosphorous imaging) are suitable.
[0028] All measurement data should be digitally recorded and
stored. The ratios of the signal intensities of test and reference
nucleic acids can then be calculated utilizing suitable
software.
EXAMPLES FOR APPLICATIONS
[0029] Important applications are in the area of clinical genetics,
tumor diagnostic, clinical pathology, the analysis of animal models
for genetic diseases including tumors and in breeding research.
[0030] Target nucleic acids for the matrix are selected in
accordance with diagnostic requirements. If, for a particular
diagnostic problem, the possible chromosomalin problems are known,
a matrix with target nucleic acids can be prepared which are chosen
selectively for the particular detection that is for the exclusion
of these specific imbalances. (See example 3 below). For other
objectives however, it is desirable to provide for as broad as
possible an analysis of the genome with regard to unknown
imbalances. This may be achieved for example by splitting the whole
genome into a series of target nucleic acids. The resolution
capability and the sensitivity of such a CNH test is then
determined by the number and the genomic distribution of the target
nucleic acids (see example 2 below). In order to achieve for
example the resolution capability of a cytogenetic banding analysis
with 400 or, respectively, 800 chromosome bands per haploidemic
chromosome set each band on the matrix should be represented by a
suitable target nucleic acid designated below as "400" or
respectively, 800 band matrix. With such a matrix losses and gains
of chromasomal regions on the so given resolution level could be
determined which corresponds to the achievable resolution
capability of CGH on metaphase chromosomes.
[0031] If necessary various matrices with different resolution
capabilities can be sequentially tested. If for example the gain or
loss of a particular chromosome segment is recognized on normal
chromosomes or a 400 band matrix, in a second step a matrix can be
used by which the breaking points of the imbalanced region can be
more accurately determined. For this matrix, target nucleic acids
are used which characterize the defined subregion of the earlier
identified chromosome segment. (Example 3).
Example 1
[0032] Screening of numerical chromosome aberrations. For this
purpose, 24 target DNAs are required which represent the 24
different human chromosomes. They are combined in accordance with
the diagnostic requirements (see below). The selection of target
DNAs may include DNA of sorted human chromosomes; DNA of somatic
hybrid cells each of which contains a human chromosome
(monochromatic hybrid cells); DNA amplification products of sorted
human chromosomes or monochromatic hybrid cells, pools of cloned,
chromosome-specific fragments such as YACs, P.sub.1-clones, cosmids
or corresponding contigs of such samples. Instead of DNAs, sorted
chromosomes or microorganisms which contain corresponding target
nucleic acids could be directly applied to the matrix (see
above).
[0033] Possible Applications
[0034] a) Prenatal screening of embryonic cells for numeric
changes. The most important numeric changes happen with respect to
the chromosomes 13, 18, 21, X and Y. Accordingly, in this case, the
matrix contains the target-DNAs of the five chromosomes referred
to. If, for ethical and legal reasons, a screening of the sex
chromosomes is to be excluded, then target-DNAs for only the
chromosomes 13, 18 and 21 would be applied.
[0035] b) Screening for hyperploids in patients with acute
lymphatic leukemia since hyperploids with n>50 have a favorable
clinical prognosis. In this case it appears to be appropriate to
apply target DNAs for all 24 human chromosomes.
[0036] c) Screening for tumors in which numeric aberrations play a
role such as chromophobic kidney cell carcinomas or bladder
carcinoma. Here too matrices to which all 24 target DNAs have been
applied could be used (which would appear to be particularly
suitable for bladder carcinomas) or to which target DNAs of the
aberrations relevant in connection with the particular tumor
entities (for example, chromophobic kidney cell carcinomas) have
been applied.
Example 2
[0037] Universal screening of unknown partial chromosome
imbalances. For this, target DNAs are required which represent
various sections of the human chromosomes. In analogy to present
molecular-biological methods of the analysis of genomic losses
("loss of heterozygosity LOH") matrices with 42 target DNAs can be
used in order to represent all the relevant chromosome arms: 1p,
1q, 2p, 2q, 3p, 3q, 4p, 5p, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 10p,
10q, 9q, 10p, 10q, 11p, 11q, 12p, 12q, 13q, 14q, 15q, 16p, 16q,
17p, 17q, 18p, 18q, 19p, 19q, 20p, 20q, 21q, 22q, Yq.
[0038] With higher resolution requirements more complex matrixes
can be employed such as the "400 or 800 band matrices described
above.
[0039] Possible Applications:
[0040] a) Screening of patients for unknown structural chromosome
aberrations b) Screening of any tumors for unknown chromosomal
imbalances. This set up is important especially in the tumor
biological research since, for many tumors, the diagnostically and
prognastically relevant genomic imbalances are presently not
identified.
Example 3
[0041] High resolution screening of certain chromosome sections for
genomic imbalances. In this case matrices are made which have
target DNAs only for selected chromosome sections and which are
concerned with a specific diagnostic objective.
[0042] Possible Applications:
[0043] a) For genetic counseling of families with reciprocal
translations, it is important to know whether genetic imbalances
have developed in the areas of the chromosomal breaking points. For
such an analysis, a matrix with high resolution can be prepared
which includes target DNAs which are mapped in the breaking point
regions in question.
[0044] b) For a Carrier-diagnosis of x-chromosomal recessive
diseases such as the Duchenne's muscular dystrophy a matrix can be
prepared which contains target DNAs for sections of the respective
gene.
Example 4
[0045] Screening for genomic imbalances of tumor-relevant genes.
For this, target-DNAs are required which represent well known
proto-onkogenes, tumor suppressor genes or other genes which are
relevant for the growth and the metastasis of a tumor.
[0046] Possible Applications:
[0047] a) The proof for the amplification of onkogenes with
prognostic relevance such as N-myc amplification in the
neuroblastoma.
[0048] b) The proof for the detection of tumor suppressor genes
with prognastic relevance such as the deletion in 1 p36 of
neuroblastoma.
Example 5
[0049] Screening for over- or under-expression of certain genes. In
this connection target nucleic acids are required which contain
coded sequences of selected genes. For this, in addition to the
matrices described in example 4, matrices with RNAs or cDNAs of the
genes may be used. As test nucleic acid, complete RNA from a cell
population to be tested is isolated; as reference nucleic acid the
complete RNA of a suitable control cell population with normal
expression of the relevant genes may be used.
[0050] Possible Applications:
[0051] With a genomic amplification of N-myo (see example 4a), a
quantitative determination of the actual over-expression can be
obtained with this test.
Example 6
[0052] The examples given above for human diseases can be utilized
in an analog manner for animal models with regard to the same
diseases. It requires the preparation of matrices whose target
nucleic acids are derived from the same species or have a
conservation which is sufficiently evolutionary for the purpose of
a CNH test.
[0053] Possible Applications:
[0054] In many animal models for specific tumors, it is first not
known whether the basic genetic mechanism corresponds to the tumor
occurring in humans. In this case, it can be expected that the
results of the CNH tests for the human and the animal tumor
correspond when a test is made for tumor-relevant genes (see
example 4) or an expression analysis (see example 5) is
performed.
Example 7
[0055] With the preparation of transgenic organisms, CNH tests with
matrices can be developed which contain target-nucleic acids of the
transferred genes. With these tests, it is possible to
quantitatively determine the numbers of copies of the transferred
genes and the expression in the receiver organism.
[0056] Possible Applications:
[0057] a) Analysis of transgenic animals with corresponding mutated
tumor relevant genes.
[0058] b) Breeding of animals and growing of plants with changed
properties.
* * * * *