Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-14
2001-08-21
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024320, C536S024330, C536S023100, C536S024100
Reexamination Certificate
active
06277577
ABSTRACT:
The invention relates to nucleic acid probes derived from the spacer region between the ribosomal ribonucleic acid (rRNA) gene, particularly between the 16S and 23S rRNA genes, to be used for the specific detection of non-viral organisms in a biological sample by a hybridization procedure.
Although much progress has been made in the last decade, for many microorganisms the diagnostic procedures currently in use are still laborious, nonsensitive and aspecific. Many of these pitfalls can be overcome by using nucleic acid probes. These nucleic acid probes can, for instance, be total genomic deoxyribonucleic acid (DNA), plasmids, riboprobes or synthetic oligonucleotides and these probes may target the genomic DNA or messenger or stable RNA species present in biological samples. Although not necessary, the use of synthetic oligonucleotides is preferred. Oligonucleotides can be rapidly synthesized in large amounts using chemical methods, have a long shelf-life, and are easily purified and labeled.
For a reliable diagnosis of microorganisms using DNA-probe technology the probes used should be highly specific (i.e. they should not cross-react with nucleic acids from other organisms) and highly sensitive (i.e. most, if not all, strains of the organism to be detected should react with the probe). Hence, the preferred target sequences should have the following characteristics:
(i) The sequence should be present in the genome of each strain of the organism concerned.
(ii) The evolutionary diversity of the sequence should be such that, on the one hand, there is sufficient sequence-diversity to allow differentiation of the species concerned from other closely related species and, on the other hand, sufficient sequence-conservation to allow the detection of all strains of the species concerned with the probe used.
Species-specific probes have been described for a large number of organisms. For a recent review see Tenover, Clin. Microbiol. Rev. 1:82-101, 1988.
However, it is not obvious from which gene in the genome that specific probe sequences can be derived. In probe development often large selection procedures have to be followed to obtain fragments which at last turn out to be specific for the organism under investigation (Korolik et al., J. Gen. Microbiol. 134:521-529, 1988; Grimont et al., J. Clin. Microbiol. 21:431-437, 1985; Welcher et al., Nucl. Acids Res. 14:10027-10044, 1986; Donegan et al., Mol. Cell. Probes 3:13-26, 1989; Beaulieu and ROY, Abstract nr D249, Abstracts of the Annual Meeting of the American Society for Microbiology, 1989). Most often the function or identity of the gene from which the specific fragment derives is not known and the screening procedure has to be blindly repeated each time another specific probe is wanted. The precise identification of a gene which meets the criteria listed above and which is ubiquitously present would obviate the need for time-consuming and tedious selections.
The 16S or 23S rRNA genes are quite often used for probe development since sequences can easily be obtained using described methods and it is known that variable regions exist within these highly conserved genes which can be used for species-specific detection. However, for certain organisms it may not be possible to derive highly specific and sensitive probes from the 16S and 23S rRNA genes, for instance, because their evolutionary nucleic acid sequence conservation is too high. Another consequence of the conserved character of these genes is that the differentiation of two organisms is often based on one or a few mismatches only in the target sequence which puts constraints on the stringency of the hybridization. A slight deviation from these conditions may result in misidentification.
Therefore the characterization of a ubiquitous gene which allows the development of species-specific probes for most organisms including those for which it was not possible to infer specific probes from the 16S and 23S rRNA genes, and which preferably have a broader stringency-range, would be extremely advantageous.
Each cellular organism possesses ribosomal RNA cistrons since its transcripts are essential for the function of ribosomes and the synthesis of proteins. In general the genes are present in multiple copies in the genome. In eubacteria the 16S rRNA gene [also called small subunit rRNA (srRNA)] is found at the 5′ end of the rRNA cistron, followed by the 23S rRNA [also called large subunit rRNA(lrRNA)]. The 5S rRNA gene is located at the 3′ end of the cistron. The 16S, 23S and 5S genes are separated by spacer regions in which transfer RNA (tRNA) genes and signal sequences involved in post-transcriptional processing may be found. At first the rRNA cistron is transcribed as one precursor RNA molecule. This primary transcript is further processed by endo- and exoribonucleases to its mature products. As a consequence, spacer region sequences are not exclusively present in the genome of the organism but also in precursor RNA molecules and processing products. The structure and processing of eubacterial rRNA cistrons is discussed in detail in the following reference: Gegenheimer and Apirion, Microbiol. Rev. 45:502-541, 1981.
The situation in nuclear genomes of eukaryotes somewhat differs in that a 5.8S RNA gene is located between the srRNA and lrRNA and 5S rRNA genes are arranged in separate long tandem arrays (Perry, Annu. Rev. Biochem. 45:605-629, 1976; Long and Dawid, Annu. Rev. Biochem. 49:727-764, 1980.). However, rRNA cistrons in the mitochondria or chloroplasts of eukaryotic organisms are prokaryotic in nature (Borst and Grivell, Nature 290:443-444, 1981).
The nucleic acid sequence of the spacer region of only a very limited number of eukaryotic or prokaryotic organisms is available from the literature (e.g. Young et al., J. Biol. Chem. 254:3264-3271, 1979; and Martens et al., System. Appl. Microbiol. 9:224-230, 1987.). From these data no reliable estimation of the nucleic acid sequence conservation can be made and consequently nothing can be concluded concerning the suitability of the spacer region for the selection of specific probes.
More precisely, concerning prokaryotes, hybridization probes derived from the spacer region between the 16S and 23S rRNA genes for the detection of microorganisms in a biological sample have not yet been described. Neither are they known for the corresponding spacer region between the small and large subunit rRNA genes of eukaryotes.
As far as eukaryotes are concerned, the use of a cloned fragment from a ribosomal gene spacer has been described in a taxonomical study on Leishmania (Ramirez and Guevara, Mol. Bioch. Parasitol. 22:177-183, 1987). However, the region used as well as the approach of the study are of no help to the man skilled in the art, for using a probe derived from the spacer region between the small rRNA and large rRNA genes, particularly for the following reasons:
(i) the ribosomal genes spacer used by Ramirez and Guevara is not the spacer region between the srRNA and lrRNA, but refers to the sequence present between two adjacent rRNA cistrons; such spacers are only found in eukaryotes between repeating units of rRNA cistrons and are not related to the internal spacer in between the srRNA and lrRNA genes;
(ii) the differentiation between Leishmania taxa using the gene spacer fragment is achieved by comparing restriction fragment patterns; the fragment used is not specific.
Hence, differentiation with the fragment using a simple hybridization protocol without resorting to Southern blot analysis is not possible.
No evidence is presented that highly specific probes can be found in that ribosomal gene spacer.
Thus, the aim of the invention is to provide species-specific probes derived from the spacer region between rRNA genes for a particular organism such as a bacterial species.
Another object of the invention is to provide DNA probes derived from the 16S-23S rRNA spacer region for the detection of
Neisseria gonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis, Haemophilus ducreyi, Haemophilus infl
Rossau Rudi
Van Heuverswyn Hugo
Merchant & Gould P.C.
Myers Carla J.
N.V. Innogenetics S.A.
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