Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-07-07
2001-07-24
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S501000, C536S025300
Reexamination Certificate
active
06265156
ABSTRACT:
This invention is concerned with improvements in and relating to hybridization histochemistry. The invention is particularly concerned with, although not limited to, improved techniques for the application of hybridization histochemistry in medical diagnosis and research.
The procedure of in situ hybridization, termed “hybridization histochemistry”, has been developed to locate in specially prepared whole sections of tissue those areas which contain specific mamqA populations, the presence of which would indicate that a certain gene is “switched on”, and production of a specific protein or peptide is, therefore, highly probable.
Hybridization between complementary strands of nucleic acids has become one of the powerful tools of molecular biology. The method stems from the fact that two complementary strands in a DNA double helix or a DNA/RNA duplex can be separated by denaturation and then reannealed (hybridized) back together under conditions where the hydrogen bonding of base pairs is favoured. One of the most exploited applications of hybridization technique has been in situ procedures where one of the annealing strands has been immobilized. Much of this work is based on the use of nitrocellulose to immobilize the DNA. Often the complementary labelled strand is added as soluble, radioactively-labelled probe which after hybridization and removal of the unbound probe, can be localized and semi-quantitated by autoradiography.
We have recently shown that these procedures can be applied to sections of tissue, by the treatment of cells or tissue sections so as to immobilize and protect naturally occurring DNA or RNA for in situ tissue hybridization while at the same time retaining sufficient cellular morphology for accurate histological location. These in situ hybridization techniques, which use radiolabeled cloned cDNAs, have been successfully employed to localize endorphin, GH, relaxin, and calcitonin mRNAs in fixed tissue slices. (See: Hudson, P., Penschow, J., Shine, J., Pyan, G., Niall, H. and Coghlan, J. Hybridization Histochemistry: Use of recombinant DNA as a “homing probe” for tissue localization of specific mRNA populations.
Endocrinology,
1981, 108: 353-356. Jacobs, J., Simpson, E., Penschow, J. P., Hudson, P., Coghlan, J. and Niall, H. Characterization and location of calcitonin mPNA in rat thyroid.
Endocrinology,
1983, 113: 1616-1622).
The basis of this technique is the incubation of a radioactively-labelled recombinant cDNA probe with a carefully prepared section of tissue. After appropriate washing the tissue is dried and autoradiography is used to identify specific cell populations or tissue regions binding the probe. The principle is thus similar to the widely used immunohistochemical procedures based upon binding of fluorescent, radioactive or peroxidase labelled antibodies.
The cDNA probe is obtained by standard cloning techniques to obtain a double-stranded copy of the target mRNA. The double-stranded copy is denatured to produce single-stranded cDNA from which labelled copy probe is made.
It was surprising that this approach has not, to our knowledge, been previously established because recombinant cDNA probes have been available for several years. There are a number of published studies which used partially purified rather than recombinant cDNA probes. This is an important distinction because recombinant probes with their absolute homogeneity guaranteed through the cloning procedure used to produce them, provide a degree of specificity of labelling which cannot be matched by any partially purified probe enriched for a particular molecular species. More recently there have been other reports on the use of cDNA probes either radioactively labelled or using fluorescent, enzymic or other labels of the hybridization sites.
Our previous work has shown clearly that it is possible where a DNA probe is available to identify which tissues secrete a particular protein. Biological applications of the techniques seem to be limited only by the specificity and type of probe which is available.
Until recently most of the work on hybridization histochemistry has been concerned with the use of probes of recombinant cDNA derived from natural mRNA or DNA. This has limited the application of the technique to cases where the natural RNA or DNA was available, e.g., had been or could be cloned from a natural source. This has naturally limited the scope of the method, as in many potential areas of application, the required RNA or DNA was not available and/or its structure was unknown.
The present invention is based on the substitution of synthetic oligonucleotide probes for cDNA obtained from or via natural sources. We have found that comparatively short probes, from about 10 to 100 nucleotides, preferably about 20 to 40 nucleotides, are usually of sufficiently unique structure to provide the necessary selectivity.
The synthesis of oligonucleotide sequences containing up to about 100 nucleotides is now readily achieved using known equipment and techniques.
Furthermore, in cases where the nucleotide sequence is not known but where information on the structure of a peptide or protein is available, it is now possible to predict with considerable certainty the DNA or RNA sequence which codes for that peptide or protein. Computer programs now available enable the redundancies in the genetic code to be resolved by predicting the most probable nucleotide sequence (for any given species) where any ambiguity exists.
Thus once the structure of a particular peptide or protein is known or can be inferred with reasonable certainty, the corresponding mRNA structure can be predicted, and a matching or complementary oligonucleotide probe can then be constructed and used to search for the natural mRNA population by the technique of hybridization histochemistry.
It is usually not necessary to know the complete amino acid sequence as useful oligonucleotide probes may be constructed from partial sequences of as few as 7 to 10 amino acids.
This approach can be used to confirm and extend studies indicating that many peptide or protein hormones are made in multiple sites (brain, gut, placenta). Biosynthesis can be distinguished from storage, and estimates of mRNA turnover made. Moreover, within a heterogeneous individual tissue (whether normal or neoplastic) it is possible to identify which cell types make a particular known product. With further increases in resolution, it should be possible to study the subcellular localization of mRNA. Probes complementary to certain non-coding regions of chromosomal DNA (e.g. to intervening sequences) might also enable studies of the location of the initial mRNA transcript (pre mRNA) and the fate of the excised segments. Specific cDNA probes can be used to detect either viral RNA/DNA or virus-specific mRNA in infected tissues. This technique could be particularly useful when a particular virus is difficult to grow in culture. This new approach to hybridization histochemistry will also prove especially useful in clinical diagnosis.
The general approach described above can also provide a method for detecting mRNA species or DNA in plant cells and tissues, in particular the detection of the state of activity of specific plant genes and the detection of plant pathogens such as, for example, plant viruses, fungi and viroids responsible for plant diseases of economic importance.
According to one aspect of the present invention there is provided a method for determining the presence and location in animal or plant tissue of a specific polynucleotide population which comprises:
(a) preparing a section of the tissue to be examined;
(b) contacting the tissue section under hybridization conditions with a synthetic, labelled oligonucleotide probe which is complementary to a representative portion of the target polynucleotide;
(c) removing unhybridized probe material from the tissue section; and
(d) detecting or identifying the locations in the tissue section where labelling by hybridization of the labelled probe has occurred.
Synthesis of oligonucleotide probes may be carrie
Coghlan John P.
Niall Hugh D.
Penschow Jennifer D.
Tregear Geoffrey W.
Howard Florey Institute of Experimental Physiology and Medicine
Marschel Ardin H.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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