Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-03
2001-05-22
Myers, Carla J. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024100, C536S024300, C536S024320, C536S024330, C435S810000
Reexamination Certificate
active
06235484
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid detection systems. More specifically, the invention relates to polynucleotide probes having binding specificity for rRNA or rDNA of the subset of Gram-positive bacteria known as the “Actinomycetes.”
BACKGROUND OF THE INVENTION
The Actinomycetes, or the “High (G+C)” subset of Gram-positive (Gram
(+)
) bacteria, are a distinct evolutionary lineage within the eubacteria. The Actinomycetes exhibit highly unusual phenotypic features as a reflection of a characteristically high mutation rate. Many members of this group of bacteria produce antibiotics and are commonly found in soil. Actinomycetes are responsible for a variety of significant animal diseases including tuberculosis, leprosy, diphtheria and periodontal diseases. Notably, immunodeficient individuals are particularly susceptible to infection by the
Mycobacteria avium, Mycobacteria intracellulare
, and
Mycobacteria scrofulaceum
, all of which are individual species among the Actinomycetes. Additionally, the Actinomycetes are responsible for a variety of economically important plant diseases.
It is well established that two single strands of deoxyribonucleic acid (“DNA”) or ribonucleic acid (“RNA”) can associate or “hybridize” with one another to form a double-stranded structure having two strands held together by hydrogen bonds between complementary base pairs. The individual strands of nucleic acid are formed from nucleotides that comprise the bases: adenine (A), cytosine (C), thymine (T), guanine (G), uracil (U) and inosine (I). In the double helical structure of nucleic acids, the base adenine hydrogen bonds with the base thymine or uracil, the base guanine hydrogen bonds with the base cytosine and the base inosine hydrogen bonds with adenine, cytosine or uracil. At any point along the chain, therefore, one may find the classical “Watson-Crick” base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may also find A:G, G:U and other “wobble” or mismatched base pairs in addition to the traditional (“canonical”) base pairs.
A double-stranded nucleic acid hybrid will result if a first single-stranded polynucleotide is contacted under hybridization-promoting conditions with a second single-stranded polynucleotide having a sufficient number of contiguous bases complementary to the sequence of the first polynucleotide. DNA/DNA, RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.
Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with the nucleic acid sequence that is to be detected (“target sequence”). Probes commonly are labeled with a detectable moiety such as a radioisotope, an antigen or a chemiluminescent moiety.
Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and 5,681,698. These references also describe methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These procedures require probes that are sufficiently complementary to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. According to the method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and then incubated under specified hybridization conditions. Conventionally, but not necessarily, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and quantitate the amount of labeled probe that has formed duplex structures in order to detect the presence of rRNA contained in the test sample.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding homologs of the procaryotic 5S, 16S and 23S rRNA molecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA which are substantially similar to the prokaryotic molecules. Probes for detecting specifically targeted rRNA subsequences in particular organisms or groups of organisms in a sample have been described previously. These highly specific probe sequences advantageously do not cross react with nucleic acids from any other bacterial species or infectious agent under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect the Actinomycetes in a highly specific manner.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an oligonucleotide probe that specifically hybridizes under high stringency hybridization conditions to a nucleic acid target region characteristic of Actinomycetes bacteria to form a detectable probe:target duplex. This target region corresponds to
E. coli
rRNA nucleotide positions 1986-2064. The invented oligonucleotide probe has a length of up to 100 nucleotides and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. In certain embodiments, the oligonucleotide probe includes at least 25 contiguous nucleotides, and may include at least 29 contiguous nucleotides contained within the sequence of SEQ ID NO:10. The high stringency hybridization condition may be provided either by 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate and 1 mM each of EDTA and EGTA, or by 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. The oligonucleotide probe may be made of DNA, but also may include at least one nucleotide analog. For example, the probe may include at least one nucleotide analog that has a methoxy group at the 2′ position of a ribose moiety. In one embodiment the invented oligonucleotide probe has a sequence that is any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, or SEQ ID NO:3 or the complement thereof. In a preferred embodiment, the sequence of the oligonucleotide is given by SEQ ID NO:2 or SEQ ID NO:3, and the oligonucleotide is a helper oligonucleotide. Any of the disclosed oligonucleotides can include a detectable label. Particular examples of detectable labels include chemiluminescent labels and radiolabels. In another preferred embodiment, the oligonucleotide has a sequence given by SEQ ID NO:1. This oligonucleotide is particularly useful as a hybridization probe, and may include a detectable label. A highly preferred detectable label for the hybridization probe is an acridinium ester.
Another aspect of the present invention relates to a probe composition for detecting nucleic acids of Actinomycetes bacteria. This composition includes an oligonucleotide probe that hybridizes under a high stringency condition to an Actinomycetes nucleic acid target region corresponding to
E. coli
23S rRNA nucleotide positions 1986-2064 to form a detectable target:probe duplex. This oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO: 10 or the complement thereof. Under high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids present in
Corynebacterium aquaticum, Corynebacterium jeikieum, Corynebacterium xerosis, Micrococcus luteus, Propionibacterium acnes, Mycobacterium chelonae, Mycobacterium terrae, Mycobacterium intracellulare, Mycobacterium simiae, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium smegatis, Mycobacteriumfortuitum, Mycobacterium gastri, Mycobacterium xenopi, Mycobacterium marinum
and
Mycobacterium phlei
. It is preferred that the oligonucleotide probe have a length of up to 100 nucleotide bases and include at least 25 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. In certain embodiments, the invented oligonucleotide probe is made of DNA. Examples of useful high stringency hybridization conditions are alternatively provided by 0.48 M sodium phospha
Gordon Patricia
Hogan James J.
Gen-Probe Incorporated
Gilly Michael J.
Myers Carla J.
Spiegler Alexander H.
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