Abundant, well distributed and hyperpolymorphic simple sequence repeats in prokaryote genomes and use of same for prokaryote classification and typing
Inactive Publication Date: 2001-11-27
TECHNION RES & DEV FOUND LTD
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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.
However, these assays do not distinguish among the various members of other serogroups.
1996), but mostly have limitations including lengthy post-amplification detection protocols or lack of template quantification.
In contrast, polymorphisms were not observed at SSR loci with two or more nucleotide core sequences, however, this may be due to the small sample size employed.
High mutation rate at SSRs result when the DNA repair system is non-functional.
Hence, it is believed that monomorphic SSR sites are under strong selection, which will not tolerate mutations in these loci.
The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37.degree. C.).
While the 3SR/NASBA, and Q.beta. systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., >55.degree. C.).
Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes.
Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification.
Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.
Traditional methods of direct detection including Northern and Southern blotting and RNase protection assays usually require the use of radioactivity and are not amenable to automation.
While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
However, specialized equipment and highly trained personnel are required, and the method is too labor-intense and expensive to be practical and effective in the clinical setting.
However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.
This technique is extremely sensitive to variations in gel composition and temperature.
A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
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Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturers' specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). The manual is hereinafter referred to as "Sambrook". Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenien
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Abstract
A method is provided for classifying or typing a prokaryote to a class or a type. The method is effected by 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. Compounds and articles of manufacture are provided for effecting the method.
Description
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