Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-27
2001-11-27
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06322985
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to classification and typing of prokaryotes and, more particularly, to abundant, well distributed and hyperpolymorphic simple sequence repeats in prokaryote genomes and the use of same for prokaryote classification and typing.
Simple sequence repeats (SSRs) are a class of short sequences, usually of 1-6 nucleotides, that are tandemly (i.e., head to tail) repeated from two or three up to a few dozen times at a locus (Vogt 1990). SSRs long have been known to be distributed throughout the genomes of eukaryotes and to be highly polymorphic (Tautz 1989, Weber 1990, Kashi et al. 1990). Polymorphisms arise primarily through slipped-strand mispairing during DNA replication (Strand et al. 1994, Tautz and Schlotterer 1994). There is accumulating evidence that SSRs serve a functional role, affecting gene expression (Kunzler et al. 1995, Kashi et al. 1997, King et al. 1997, Kashi and Soller 1998).
The sequencing of complete genomes of many prokaryotes presented the opportunity to screen such genomes for the existence of SSRs (Field and Wills 1996, 1998), revealing arrays not detected in earlier studies. Recent publication of the complete genome sequence for
Escherichia coli
(Blattner et al. 1997) provides the basis for characterization of its SSR arrays, both at a gross genomic level and at particular SSR loci.
Present-day approaches for typing of prokaryotes include growth in selective media, binding of specific antibodies, and amplification of DNA using the polymerase chain reaction. For example, conventional methods for detection of
E. coli
(Vanderzant and Spittstoesser 1992) include enrichment and isolation with selective or indicator media, such as
E. coli
(EC) broth, lauryl sulfate tryptose 4-methylumbeliferyl-&bgr;-D-glucaronic acid broth, eosin methylene blue agar, and McConkey sorbitol agar. Procedures based on use of such media lead to identification of
E. coli
in a sample and estimation of number, but lack the ability to distinguish among
E. coli
strains. Hence, the entire process of strain identification remains difficult and time-consuming. Recent methods for identification of
E. coli
strains use antibodies or nucleic acid sequences that uniquely bind to a particular strain or group of strains. Several methods for immunological detection br biochemical identification of the toxin-producing
E. coli
strain 0157:H7 have been described (Farmer and Davis 1985, March and Ratnam 1986, Kleanthous et al, 1988, Smith and Scotland 1988, Todd et al. 1988, Karmali 1989, Padbye and Doyle 1991, Tyler et al. 1991). However, these assays do not distinguish among the various members of other serogroups. DNA amplification-based assays have been reported (Karch and Meyers 1989, Pollard et al. 1990, Johnson et al 1990, Johnson et al. 1991, Jackson 1991, Yu and Kaper 1992, Witham et al. 1996), but mostly have limitations including lengthy post-amplification detection protocols or lack of template quantification.
DNA sequence determination, on the other hand, is much more simple and accurate.
There is thus a widely recognized need for, and it would be highly advantageous to have, a simple and rapid DNA sequence based technique for the classification and typing of prokaryotes.
While conceiving the present invention it was assumed that prokaryotes SSRs might be polymorphic and that such polymorphism might be class and type correlated and, if indeed exists, could be used to provide a simple tool for the presently labor-intensive and complicated task of classification and typing of prokaryotes.
While reducing the present invention to practice, length polymorphism was shown at two mononucleotide SSR loci in
E. coli
. The existence of thousands of SSR arrays in
E. coli
and in a wide range of other prokaryotes that should exhibit hypervariability is shown as well. Interestingly, these SSR sites exhibit an upper size limit of 12 bp, suggesting selective mechanisms that might impose this size limit.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of classifying or typing a prokaryote to a class or a type, the method comprising the step of characterizing at least one polymorphic simple sequence repeat locus in a genome of the prokaryote and, based on a characterization of the polymorphic simple sequence repeat, classifying or typing the prokaryote to a class or a type.
According to another aspect of the present invention there is provided a pair of polymerase chain reaction primers having a sequence adapted for exponential amplification of a polymorphic simple sequence repeat locus in a genome of a prokaryote.
According to yet another aspect of the present invention there is provided a polymerase chain reaction product derived by amplifying a portion of the genome using the pair of polymerase chain reaction primers described above.
According to still another aspect of the present invention there is provided an allele specific oligonucleotide comprising a sequence of nucleotides adapted for effectively hybridizing only with a specific simple sequence repeat of a polymorphic simple sequence repeat locus in a genome of a prokaryote, under stringent allele specific oligonucleotide hybridization conditions of (i) a hybridization solution of 2×standard sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS); (ii) a hybridization temperature of from 42° C. to Tm −5° C. for 30 minutes to overnight, wherein Tm is estimated as 2×(the number of A plus T residues)+4×(the number of G plus C residues); and (iii) post hybridization washes with 0.75×SSC and 0.1% SDS at a temperature from 42° C. to Tm −5° C. For further details see Bult, C. J., et al., which is incorporated by reference as if fully set forth herein.
According to still an additional aspect of the present invention there is provided a DNA chip comprising a surface and a plurality of allele specific oligonucleotides attached thereto, each of the plurality of allele specific oligonucleotides including a sequence of nucleotides adapted for effectively hybridizing only with a specific simple sequence repeat of a polymorphic simple sequence repeat locus in a genome of a prokaryote, under stringent hybridization conditions as described above. Preferably, the sequence of nucleotides is perfectly complementary to the specific simple sequence repeat.
According to an additional aspect of the present invention there is provided a hybrid of the allele specific oligonucleotide described above and the specific simple sequence repeat.
According to yet additional aspect of the present invention there is provided a primer having a sequence adapted for amplification of a polymorphic simple sequence repeat locus in a genome of a prokaryote.
According to further features in preferred embodiments of the invention described below, characterizing the at least one polymorphic simple sequence repeat locus in the genome of the prokaryote is effected by an allele specific oligonucleotide hybridization.
According to still further features in the described preferred embodiments characterizing the at least one polymorphic simple sequence repeat locus in the genome of the prokaryote is effected by a polymerase chain reaction.
According to still further features in the described preferred embodiments characterizing the at least one polymorphic simple sequence repeat locus in the genome of the prokaryote is effected by a sequencing reaction.
According to still further features in the described preferred embodiments characterizing the at least one polymorphic simple sequence repeat locus in the genome of the prokaryote is effected by a heteroduplex hybridization reaction.
According to still further features in the described preferred embodiments characterizing the at least one polymorphic simple sequence repeat locus in the genome of the prokaryote is effected by single strand conformational polymorphism.
According to still further features in the described preferred embodiments characterizing the at least one pol
Cohen Cyril
Eitan Yuval
Gur-Arie Riva
Hallerman Eric
Kashi Yechezkel
Fredman Jeffrey
Technion Research and Development Foundation Ltd.
LandOfFree
Abundant, well distributed and hyperpolymorphic simple... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Abundant, well distributed and hyperpolymorphic simple..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Abundant, well distributed and hyperpolymorphic simple... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2583932