WO1999051961A2

WO1999051961A2 - Improved method for chromosome-specific staining

Info

Publication number: WO1999051961A2
Application number: PCT/US1999/007544
Authority: WO
Inventors: Jane M. Scalzi Martin; Joseph R. Bertino
Original assignee: Scalzi Martin Jane M; Bertino Joseph R
Priority date: 1998-04-06
Filing date: 1999-04-06
Publication date: 1999-10-14
Also published as: AU3384999A

Abstract

The present invention provides a method of staining a chromosome or chromosome region comprising: a) contacting labeled nucleic acid with a region of chromosomal DNA of interest, wherein said labeled nucleic acid comprises fragments substantially complementary to nucleic acid segments within the chromosomal DNA, including both unique sequences of DNA and repetitive sequences; b) hybridizing said labeled nucleic acid to target chromosomes in the absence of blocking DNA allowing both unique sequence and repetitive sequence fragments of the labeled nucleic acid to hybridize; and c) washing said target chromosomes containing bound labeled nucleic acid to differentially remove bound repetitive sequence fragments of the labeled nucleic acid while specific region labeled nucleic acid fragments remain bound to the target chromosomes allowing detection of hybridized labeled nucleic acid containing unique sequences, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization.

Description

IMPROVED METHOD FOR CHROMOSOME-SPECIFIC STAINING

BACKGROUND OF THE INVENTION

Cross-reference to Related Application This patent application claims benefit of provisional patent application U.S. Serial number 60/080,823, filed April 6, 1998, now abandoned.

Field of the Invention The present invention relates generally to the field of cytogenetics and, more particularly, to methods for identifying and classifying chromosomes. The invention is further characterized that the invented method simplifies current methods and additionally avoids the use of expensive and toxic reagents .

Description of the Related Ar Chromosome abnormalities are associated with genetic disorders, degenerative diseases and exposure to agents known to cause degenerative diseases, particularly cancer. Chromosome abnormalities can be of three general types, extra or missing individual chromosomes, extra or missing portions of a chromosome or chromosomal rearrangements. Detectable chromosomal abnormalities occur with a frequency of one in every 250 human births. Abnormalities that involve deletions or additions of chromosomal material alter the gene balance of an organism and generally lead to fetal death or to serious mental and or physical defects. Prior art methods and compositions for staining chromosomes comprise heterogeneous mixtures of labeled nucleic acid fragments having substantial portions of substantially complementary base sequences to the chromosomal DNA for which specific staining is desired. The nucleic acid fragments of the heterogeneous mixtures include double stranded or single stranded RNA or DNA. Heterogeneous, in reference to the mixture of labeled nucleic acid fragments, means that the staining reagents comprise many copies each of fragments having different base compositions and/or sizes, such that application of the staining reagent to a chromosome under the appropriate molecular hybridization conditions in situ, results in a substantially uniform distribution of fragments hybridized to the chromosomal DNA by complementary base pairing. As discussed more fully below it is desirable to disable the hybridization capacity of repetitive sequences because copies occur on all or many of the chromosomes of a particular organism; thus, their presence reduces the chromosome specificity of the staining reagents. Hybridization capacity can be disabled in several ways, e.g., selective removal or screening of repetitive sequences from chromosome specific DNA or selective blocking of repetitive sequences by pre-reassociation with sequence-complementary fragments. As mentioned above, it is desirable to disable the hybridization capacity of repetitive sequences by removal, block, or similar means. Repetitive sequences are distributed throughout the genome; many are not chromosome-specific. Consequently, in spite of the fact that the nucleic acid fragments are derived from isolated chromosomes or specific regions of chromosomes, the presence of repeats greatly reduces the degree of chromosome-specificity of the staining reagents, particularly in genomes containing a significant fraction of repetitive sequences, such as the human genome. Several prior art techniques are available for disabling the hybridization capacity of repetitive sequences. For example, highly repetitive DNA sequences can be removed from extracted chromosome-specific DNA by denaturing and incubating the extracted DNA against itself or against repetitive-sequence- enriched total genomic DNA on hydroxyapatite, or a similar absorbent, at low C₀t values (initial DNA concentration multiplied by hybridization time). Hydroxyapatite chromatography is a standard technique for fractionating DNA on the basis of reassociation conditions such as temperature , salt concentration . Hydroxyapatite chromatography is also useful for fractionating DNA on the basis of reassociation rate at fixed reassociative conditions, or stringencies. Fractionation based on resistance to S I nuclease can also be used to separate single stranded from double stranded DNA after incubation to particular C₀ t values. However, these procedures are disadvantageous as they are costly and require a substantial investment in time. In addition to self hybridization or hybridization against repetitive-sequence-enriched total genomic DNA, removal of repeats from fragment mixtures can also be accomplished by hybridization against immobilized high molecular weight total genomic DNA, following a procedure described by Brison et al. Briefly, the procedure removed repeats from fragment mixtures in the size range of a few tens of bases to a few hundred bases. Minimally sheared total genomic DNA is bound to diazonium cellulose, or similar support. The fragment mixture is then hybridized against the immobilized DNA to C₀t values in the range of about 1 to 100. The preferred stringency of the hybridization conditions may vary depending on the base composition of the DNA. Similar methods of repeat depletion recently have been applied specifically to chromosome staining reagents (Craig et al 1997). A preferred means for disabling hybridization capacity is selecting unique sequence nucleic acid inserts from a chromosome-specific DNA library. For example, following the method of Benton and Davis, 1997, pieces of chromosome-specific DNA are inserted into lambda gt bacteriophage or like vector. The phages are plated on agar plates containing a suitable host bacteria. DNA from the resulting phage plaques is then transferred to a nitrocellulose filter by contacting the filter to the agar plate. The filter is then treated with labeled repetitive DNA so that phage plaques containing repetitive sequence DNA can be identified. Those plaques that do not correspond to labeled spots on the nitrocellulose filter comprise clones that may contain only unique sequence DNA. Clones from these plaques are selected and amplified, radioactively labeled, and hybridized to Southern Blots of genomic DNA that has been digested with the same enzyme used to generate the inserted chromosome-specific DNA. Clones carrying unique sequence inserts are recognized as those that produce a single band or a few bands during Southern Analysis. Another method of disabling the hybridization capacity of repetitive DNA sequences within nucleic acid fragments involves blocking the repetitive sequences by pre- reassociation of the target DNA with fragments of repetitive sequence enriched DNA, or pre-reassociation of both the target DNA with repetitive-sequence-enriched DNA. The method is generally described by Sealy et al. 1985. The term pre-reassociation refers to a hybridization step involving the reassociation of unlabeled, repetitive DNA or RNA with the nucleic acid fragments of the heterogeneous mixture just prior to the in situ hybridization step, or with the target DNA, for example a cytogenetic perforation on a microscope slide, either just prior to or during the in situ hybridization step. This treatment results in nucleic acid fragments whose repetitive sequences are blocked by complementary fragments such that sufficient unique sequences regions remain free for attachment to chromosomal DNA during the m situ hybridization step. Three factors influence the staining sensitivity of a heterogeneous mixture of DNA's or RNA' s (i.e. the hybridization probe): ( 1 ) efficiency of hybridization (fraction of target DNA that can by hybridized by probe), (2) detection efficiency (i.e., that amount of visible signal that can be obtained from a given amount of hybridization probe), and (3) level of noise produced by nonspecific binding of probe or components of the detection system. The fixed chromosomes can be treated in several ways either during or after the hybridization step to reduce nonspecific binding of probe DNA. Such treatments include adding large concentrations of non-probe or "carrier" DNA to the heterogeneous mixture, using coating solutions, such as Denhardt' s solution (Biochem Biophys. Res. Commun.. Vol. 23, pp. 641-645 ( 1966)), with the heterogeneous mixture incubating for several minutes, e.g., 5-20 minutes, in denaturing solvents at a temperature 5°- 10°C above the hybridization temperature and in the case of RNA probes, mild treatment with single strand RNase (e.g., 5 to 10 micrograms per milliliter RNase in 2 X SSC at room temperature for 1 hour). Many of the whole chromosome and region specific probes utilized require the addition of competitor DNA such as C₀tl for signal specificity due to varying levels of repetitive DNA inherent in the probe. The use of chromosome in situ suppression (Lichter 1988) and similar methods (Pinket et al., 1986) utilizing competitor or blocking DNA in the probe mix has been examined. The high cost of producing probes containing repetitive DNA, particularly multi-chromosome probes which generally require large amounts of competitor DNA, has restricted the number of potential users of these tools. These issues force the development and commercialization of repeat- free fluorescence in situ hybridization probes, a labor intensive enterprise, and alternative protocols. Thus, the prior art is deficient in methodology to conveniently prevent repetitive DNA interference in specific chromosome-staining procedures. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method for chromosome-specific staining with labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA in the absence of competitor DNA. This method of hybridization and post-hybridization treatments produces specific probe signal in the absence of competitor DNA. This technique is particularly useful as these methods may be used with existing probes without requiring lengthy and costly post-probe development modifications. Furthermore, the protocol is simple and can be substituted for most standard protocols without increasing experiment time and may be generally applicable regardless of the species or method of probe production. In one embodiment of the present invention, there is provided a method of staining a chromosome or chromosome

7 regions comprising: (a) contacting labeled nucleic acid with a region of chromosomal DNA of interest, wherein said labeled nucleic acid comprises fragments substantially complementary to nucleic acid segments within the chromosomal DNA, including both unique sequences and repetitive sequences; (b) hybridizing said labeled nucleic acid to said chromosomal DNA in the absence of blocking DNA allowing both unique sequence and repetitive sequence fragments of the labeled nucleic acid to hybridize; and (c) washing said chromosomal DNA containing bound labeled nucleic acid to differentially remove bound repetitive sequence fragments of the labeled nucleic acid while specific region labeled nucleic acid fragments remain bound to the target chromosomal DNA allowing detection of hybridized labeled nucleic acid containing unique sequences, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization . Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently referred embodiments of the invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE FIGURES

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended figures. These figures form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope . Figure 1 shows a fluorescence in situ hybridization experiment using paint probes specific for human chromosome 3. Figure 1A shows the signal obtained using a standard procedure which includes C₀ tl competitor DNA and the paint probe for human chromosome 3. Figure I B shows the signal obtained using the standard procedure but excluding the C₀tl competitor DNA and the identical paint probe for human chromosome 3 as in Figure 1A. Figure IC shows the signal obtained using the method of the present invention which does not include C₀ t l competitor DNA and the identical paint probe for human chromosome 3 as in Figures 1A and IB. Figure 2 shows a fluorescence in situ hybridization experiment using paint probes specific for human chromosome 3 using the method of Weinerg et al. ( 1997), which increases the self-annealing time to 3 hours using the identical paint probe for human chromosome 3 as in Figures 1A, IB and IC u nder standard wash temperatures. The nucleic acid is labeled with the fluorescent dye Cy3 in Figure 1 and 2 , and labeled chromosomes are viewed by fluorescence microscopy. Figure 3 shows that the method of the present invention as applied to a sub-chromosomal nucleic acid probe.

9 Figure 3 A shows a human chromosome 17-specific oncogene Her2/neu probe, a four cosmid contig that targets 90Kb of genomic sequence, using essentially the hybridation/wash conditions of Figure IB , without C₀tl competition. Figure 3B shows the same probe as in Figure 3 A under conditions of the method of this invention. The nucleic acid was labeled with biotin and detected indirectly with avidin/fluorescence by standard techniques.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed towards a new method of hybridization and post-hybridization treatments to produce specific probe signal in the absence of competitor DNA for the staining of chromosomes. The methodology of the invention is superior to other suppression or non-suppression fluorescence in situ hybridization methods for several reasons. First, no post production probe modification is required. Secondly, no increase in standard experiment time is required. Thirdly, costs are reduced in the absence of competitor DNA. Fourthly, cost, stability and disposal issues are eliminated in the absence of formamide. Finally, the procedure of the invention is likely to be applicable to all species, independent of the availability of blocking DNA for the species. In accordance with the above-mentioned object there is provided a method for the staining of chromosomes in situ that obviates the need for competitor DNA or in any way removing repetitive DNA sequences from the probe. This method of the

10 present invention comprises : (a) labeling heterogeneous mixtures of nucleic acid fragments having substantially complementary base sequences to unique sequence regions of chromosomal DNA; (b) applying labeled nucleic acid fragments to chromosomes; and (c) washing selectively a repetitive sequence probe from the chromosomes. Following the hybridization of the probe DNA with the chromosomes, the method of the present invention further comprises one or a series of high stringency washes. High stringency washes can be performed at a range of temperatures (generally 65°C-72°C) and salt concentration (generally l x - O.Olx sodium citrate/sodium chloride; SSC). The optimal conditions for any given probe composition may be determined routinely by a person having ordinary skill in this art. The high stringency washes are for 1 minutes to 20 minutes or longer and may be composed of several washes at similar or increasingly dilute salt concentration (e.g. 68° C, 2xSSC for 10 minutes and repeated, followed by 68°C, O. lx SSC for 10 minutes and repeated) or changes in temperature or any suitable combination of wash conditions that achieve differential removal on non- specific repeat hybridization signals. By means of these wash conditions non-unique probe sequences are removed from the chromosomes while the unique probe sequences remain bound. Thus, the specificity of the probe sequences is revealed. The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:

11 EXAMPLE 1 Fluorescence in situ hybridization In the present invention target chromosome preparation, probe labeling and fluorescent in situ hybridization were essentially performed as described in Liechty et. al., (1995). Labeled nucleic acids used in the method of this invention contained only DOP PCR product or cosmid DNA and yeast tRNA or other non-target nucleic acid as carrier. Such probes were self- annealed for 1 hour or 3 hours at 37° C prior to application to the target chromosomes.

EXAMPLE 2 Post-Fluorescence SitU hybridization washes : chromosome paint probe Hybridization slides were washed at a range of temperatures (65-72°C) and salt concentrations ( lx - O.Olx sodium citrate/sodium chloride; SSC) to assay the optimal conditions for differential repeat removal. In this particular case the best signal to noise ratio was achieved by washing at 68° C, twice in 2xSSC and twice in O. lxSSC for 10 minutes each with agitation. Slides were rinsed in BN buffer (Liechter et al., 1995) before proceeding with signal detection or in the case of directly labeled probes, before applying counterstain. To evaluate the effects of wash solution temperature and stringency on probe specificity in the absence of DNA competition, hybridization were performed with several control reactions. Paint probes containing competitor (C°tl DNA) amounts at 50 fold the amount of labeled probe were pre-

12 annealed for one hour in accordance with most fluorescence i n situ hybridization paint procedures. These positive control reactions were post-washed at standard protocol temperatures ( 45°C) in non-formamide salt washes. Under these conditions, a paint probe for human 3 was chromosome specific (Figure 1A). The same probe prepared without C₀tl and treated under identical conditions, produced a non-specific "metaphase paint"; signal covering all chromosomes in a metaphase spread (Figure IB). When the post wash solution temperature was increased to 6 8°C, probe specificity of non-competed DNA was comparable to the standard fluorescence in situ hybridization reaction containing C°tl (Figure IC). Contrary to a recent report on non- competed probes (Weinberg et al., 1997), increasing the self- annealing time to 3 hours using these paint probes under standard wash temperature, did not result in probe specificity (Figure 2). Thus, the present invention is directed to a method of staining a chromosome or chromosome region comprising: (a) contacting labeled nucleic acid with a region of chromosomal DNA of interest, wherein said labeled nucleic acid comprises fragments substantially complementary to nucleic acid segments within the chromosomal DNA, including both unique sequences and repetitive sequences; (b) hybridizing said labeled nucleic acid to said chromosomal DNA in the absence of blocking DNA allowing both unique sequence and repetitive sequence fragments of the labeled nucleic acid to hybridize; and (c) washing said chromosomal DNA containing bound labeled nucleic acid to differentially remove bound repetitive sequence

13 fragments of the labeled nucleic acid while specific region labeled nucleic acid fragments remain bound to the target chromosomal DNA allowing detection of hybridized labeled nucleic acid containing unique sequences, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization. Preferably, the hybridizing of labeled nucleic acid to target chromosomes occurs at a temperature of from about 25° C to about 70°C, for a time of from about 30 minutes to about one week and the probe and/or carrier DNA may be incubated together prior to hybridization of the labeled nucleic acid with the chromosomal DNA. Preferably, the washing of the target chromosomes occurs by treating with a solution to differentially remove portions or fragments of the labeled nucleic acid from complimentary regions of the chromosomal DNA. This method of the present invention may be used to stain any chromosome or chromosome region, including chromosomal DNA such as human DNA, primate DNA, rodent DNA, mouse DNA and rabbit DNA. The labeled nucleic acid used in this method may comprise fragments which allow detection of extra or missing chromosomes, extra or missing portions of a chromosome, or chromosomal rearrangements. For example, the chromosomal rearrangement may be a translocation or an inversion. Preferably, the labeled nucleic acid is designed to detect a chromosomal rearrangement consistent with chromic myelogenous leukemia or the labeled nucleic acid is designed to detect aneuploidy or amplified regions of a chromosome. A representative extra chromosome is chromosome 21.

14 Preferably, the labeled nucleic acid comprises fragments complementary to the total genomic complement of chromosomes, fragments complementary to a single chromosome, fragments complementary to a subset of chromsomes, or fragments complementary to a subregion of a single chromosome. Representative fragments include the nucleic acid of normal human chromosomes 1 through 22, X and Y. In another embodiment, the chromosomal DNA is metaphase or interphase chromosomal DNA. In the method of the present invention, the repetitive segments of the labeled nucleic acid comprise segments which are substantially complementary to highly repetitive segments may be substantially complementary to satellite repetitive segments or the repetitive segments may be substantially complementary to tandem repetitive segments or repetitive segments located in regions of centromeric heterochromatin. Optionally, the repetitive segments are substantially complementary to interspersed repetitive segments. The following references were cited herein: Benton and Davis, Science, 196: 180-182 (1977); Brison et al., Molecular and Cellular Biology , 2(15): 578-587 (1982); Sealy et al., Nucleic Acid Research, 13: 1905-1922 (1985); Gall and Pardue, Methods in Enzymology 21 : 470-480 (1981); Henderson, International Review of Cytology, 76: 1-46 ( 1982); Angerer, et. al., "In Situ Hybridization to Cellular RNAs," in Genetic Engineering: Principles and Methods (Setlow and Hollaender, Eds.) Vol. 7 pp. 43-68 Plenum Press, New York (1985); Weinberg et al., Elsevier Trends Journals: Technical Tips Online, T ADD 65, 1997;

15 Britten, RJ. and Davidson, EH. pp 3-11 , In Nucleic Acid Hybridization - A Practical Approach (B.D. James and J.J. Higgins, eds.) IRL PRess, Oxford (1985); Craig et al., Human Genet. 100: 472-476 (1997); Lichter et al. Science 247: 64- 69 (1990); Pinkel et al., Proc. Nat. Acad. Sci. USA 83: 2934- 2938 ( 1986); and Liechty et al., Mouse chromosome- specific painting probes generated from microdissected chromosomes, Mammalian Genome 6:592-594. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled int he art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein ad other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

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Claims

WHAT IS CLAIMED IS:

1 . A method of staining a chromosome o r chromosome region comprising: ( a) contacting labeled nucleic acid with a region of chromosomal DNA of interest, wherein said labeled nucleic acid comprises fragments substantially complementary to nucleic acid segments within the chromosomal DNA, including both unique sequences and repetitive sequences; (b) hybridizing said labeled nucleic acid to said chromosomal DNA in the absence of blocking DNA allowing both unique sequence and repetitive sequence fragments of the labeled nucleic acid to hybridize; and (c) washing said chromosomal DNA containing bound labeled nucleic acid to differentially remove bound repetitive sequence fragments of the labeled nucleic acid while specific region labeled nucleic acid fragments remain bound to the target chromosomal DNA allowing detection of hybridized labeled nucleic acid containing unique sequences, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization.

2. The method of claim 1 , wherein said hybridizing of labeled nucleic acid to chromosomal DNA occurs at a temperature of from about 25┬░ C to about 70┬░ C and for a time of from about 30 minutes to about one week and the nucleic acid probe and/or carrier DNA may be incubated together prior to hybridization of the labeled nucleic acid with the chromosomal

17 DNA.

3 . The method of claim 1 , wherein washing of said chromosomal DNA occurs by treating with a solution to differentially remove portions or fragments of the labeled nucleic acid from complimentary regions of the chromosomal DNA, at temperatures of approximately 50┬░C to 95┬░ C, for times of from 15 seconds to 1 hour, for one or multiple times.

4. The method of claim 1 , wherein said chromosomal DNA is human DNA.

5 . The method of claim 1 wherein said chromosomal DNA is selected from the group consisting of primate DNA, rodent DNA, mouse DNA and rabbit DNA, porcine DNA, ovine DNA and bovine DNA.

6. The method of claim 1 , wherein the labeled nucleic acid comprises fragments which allow detection of extra or missing chromosomes, extra or missing portions of a chromosome, or chromosomal rearrangements.

7 . The method of claim 6 , wherein the chromosomal rearrangement is a translocation or an inversion.

8 . The method of claim 6, wherein the labeled nucleic acid detects a chromosomal rearrangement consistent with chromic myelogenous leukemia.

9. The method of claim 6, wherein the labeled nucleic acid detects aneuploidy, or amplified segments of chromosomes .

1 0. The method of claim 6, wherein the extra chromosome is chromosome 21.

1 1 . The method of claim 1 , wherein the labeled nucleic acid comprises fragments complementary to the total genomic complement of chromsomes, fragments complementary to a single chromosome, fragments complementary to a subset of chromosomes, or fragments complementary to a subregion of a single chromosome.

12. The method of claim 11 , wherein the fragments are selected from the nucleic acid of normal human chromosomes 1 through 22, X and Y.

1 3. The method of claim 1 wherein the chromosomal DNA is metaphase or interphase chromosomal DNA.

14. The method of claim 1 , wherein the repetitive segments comprise segments which are substantially complementary to highly repetitive segments and middle repetitive segments.

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1 5 . The method of claim 14, wherein the repetitive segments are substantially complementary to satellite repetitive segments.

1 6. The method of claim 15, wherein the repetitive segments are substantially complementary to tandem repetitive segments or repetitive segments located in regions of centromeric heterochromatin.

17. The method of claim 15, wherein the repetitive segments are substantially complementary to interspersed repetitive segments.

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