Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-25
2001-06-05
Marschel, Ardin H. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S025300
Reexamination Certificate
active
06242184
ABSTRACT:
BACKGROUND OF THE INVENTION
Hybridization is a general technique in which the complementary strands of deoxyribonucleic acid (hereinafter “DNA”) molecules, ribonucleic acid (hereinafter “RNA”) molecules, and combinations of DNA and RNA are separated into single strands and then allowed to renature or reanneal into base-paired double helices. At least three major classes of hybridization are conventionally known and used: solution hybridization which disrupts the individual cells and extracts the internal nucleic acids into solution prior to hybridization; filter or blot hybridization which transfers extracted DNA (or RNA) fragments from agarose gels to filters or blotters such as cellulose nitrate or nylon for subsequent hybridization with radioactive DNA or (RNA) and then detection of hybridization by radioautography or fluorography; and in-situ hybridization which makes possible the detection and localization of specific nucleic acid sequences directly within a structurally intact cell or cellular component where extraction of nucleic acids from the cell is undesirable. Although each of these respective hybridization techniques often employ cells, tissues, and certain reagents in common, each technique is generally viewed and accepted within this art as different and completely distinguishable from any other.
In-situ hybridization is a technique which yields both molecular and morphological information about intact individual cells and cellular parts. Rather than requiring the investigator to laboriously extract DNA and/or RNA from a heterogeneous cell population, the technique permits detection of DNA and RNA in-situ within the cellular morphology and allows the investigator to identify those particular cells or cell parts which contain specific DNA or RNA sequences of interest. This technique also allows one to determine simultaneously the biochemical and/or morphological characteristics of these cells. For this reason, the in-situ hybridization methodology has direct application for many areas of biomedical and clinical research including developmental biology, cell biology, genetics, clinical diagnosis, and pathological evaluation.
Despite the potential of in-situ hybridization as a molecular analytical technique, the development of effective protocols and procedures has been largely haphazard and disjointed. Since first described in 1969 by Gall et al.,
P.N.A.S. U.S.A
., 63:378-383 (1969);
Methods in Enzymol
., 38:370-380 (1971), the in-situ hybridization approach has been directed towards two different morphological situations: the localization of specific nucleic acid sequences of interest in the cytoplasm of a cell; and the identification of specific nucleic acids within the nucleus and/or chromosomes of a cell.
The other major application of in-situ hybridization has been for the detection of specific nucleic acid sequences within individual chromosomes and genes; and for detection of extrachromosomal nucleic acids within the cellular nucleus or cytoplasm. These investigations have been widely applied for the detection of DNA sequences in polytene chromosomes and to the identification of highly reiterated DNA sequences in metaphase chromosomes. In recent years, some investigators have also been able to localize single-copy DNA sequences in relatively large chromosomal segments using radiolabeled probes and a statistical analysis of autoradiographic grain distributions. However, due to the scatter of radioactive disintegrations, the resolution of this approach is limited and localization of DNA is possible only within relatively large chromosomal segments in the metaphase nucleus. Futhermore, because localization of the sequence is not determined directly within a single cell, identification requires statistical analysis of the autoradiographic grain distribution in an many as 50 to 100 metaphase figures. Garson et al. (
Nucleic Acids Research
, Volume 15, Number 12, (1987), pp 4761-4771) and Landegent et al. (
Nature
, Volume 317, 12, (September 1985)) teach hybridization methods for detecting unique sequences using non-isotopic detection systems. Neither method demonstrated detection of a single copy sequence or unique sequence based upon the analysis of a single cell. A statistical analysis of many cells was necessary when analyzing the results. Because of these limitations, it has not been possible with prior art techniques to localize single or low copy sequences within the interphase nucleus.
The development and applications of in-situ hybridization had been largely qualitative rather than quantitative in nature; although several investigators have developed a quantitative approach using autoradiography for cells to which a probe had been hybridized methods were typically limited to testing only a very few samples at one time and were enormously time consuming and laborious procedures. Equally important, protocol parameters such as choice of fixation, the need for cell pretreatment prior to hybridization, the size of the probes utilized, the concentration of probe, and the time required for hybridization varied markedly among the different protocols published. Moreover, the then known protocols were also highly complex procedures requiring many manipulative steps, all too many of which are actually destructive to the cell by their ability to dissociate either the nucleic acid components within the cell and/or the structural morphology and overall integrity of the cell. Further, the known protocols varied extensively in their sensitivity limits, their reproducibility, and their general effectiveness.
In our copending application Ser. No. 06/790,107, filed Oct. 22, 1985, we describe a rapid in-situ hybridization method for detecting target nucelic acid sequences in morphologically intact cells. The teachings of the copending application are hereby incorporated herein by reference.
SUMMARY OF THE INVENTION
The present invention is a rapid non-isotopic method for detecting a target nucleic acid sequence in a cellular sample containing nucleic acid with a nucleic acid probe, comprising:
a) fixing said nucleic acid in said cellular sample to allow the nucleic acid probe to hybridize to the target nucleic acid sequence;
b) contacting said cellular sample with the nucleic acid probe for less than about 24 hours under conditions whereby said probe hybridizes to the target nucleic acid sequence, said probe consisting essentially of a multiplicity of non-isotopically labelled nucleic acid fragments complementary to a portion of said target nucleic acid sequence, said fragments having from about 20 to about 1000 nucleotides;
c) detecting said labelled probe.
This rapid, non-isotopic in situ hybridization method provides exceptionally high hybridization efficiency, low background, and high resolution. Indeed, it provides the capability to detect a single copy of a single nucleic acid sequence within a single cell.
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Angerer et al., Nucleic Acids Research 9(12):2819-2839, 1981.
Bauman et al., Cytometry 9:517-524, 1988.
Brandsma et al., Proc. Natl. Acad. Sci. USA 11:6851-6855, 1980.
Brigati et al., “Detection of Viral Genomes In Cultured Cells and Paraffin-Embedded Tissue Sections Using Biotin-Labeled Hybridization Probes”, Virology 126:32-50, 1983.
Capco et al., Developmental Biology 67:137-151, 1978.
Gee et al., DNA 2:157-163, 1983.
Godard et al., Histochem 65:291-300, 1980.
Gray et al., “Fluorescence Hybridization to Human Chromosome 21 Using Probes From a Charon 21 A Library”, Cytometry, Supplement 1, Abstracts 1987.
Harper
Johnson Carol Villnave
Lawrence Jeanne Bentley
Singer Robert H.
Fish & Richardson P.C.
Marschel Ardin H.
University of Massachusetts
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