Image analysis – Applications – Biomedical applications
Reexamination Certificate
2000-06-02
2004-05-18
Mariam, Daniel (Department: 2621)
Image analysis
Applications
Biomedical applications
C382S167000, C382S191000, C382S287000
Reexamination Certificate
active
06738502
ABSTRACT:
BACKGROUND OF THE INVENTION
Identification of microbes (such as bacteria, archaea and simple eucarya) using DNA and other hybridization probes has become increasingly sophisticated and accurate as probe technology and methods have improved. Probes are molecules that bind with high affinity and specificity to target molecules. Up to now, instrumentation for detecting fluorescently-labeled or other spectroscopically identifiable probes has not been able to fully exploit the capabilities of such molecules to identify in situ highly complex (i.e., genotypically or phenotypically diverse) mixtures of biological cells and viruses. Positive identification of single variant organisms in large populations of similar cells is also problematic. Fluorescent and other spectroscopically identifiable labeling methods are ultimately limited by the number of different spectral ‘fingerprints’ that can be distinguished by the imaging system that is used to measure and sort them. We have created probe sets labeled with as many as eight distinct fluorophores for simultaneous hybridization against 16S small subunit RNA and other targets. These fluorophores are all organic dyes with relatively broad absorption and fluorescence emission bands; however, newly developed inorganic fluorescent quantum dots or nanocrystals (with narrower fluorescence emission bands) can also be used. KAIROS has developed instrumentation and spectral deconvolution and sorting algorithms to increase the number of spectroscopically identifiable tags that can be simultaneously distinguished, thus enabling accurate ‘fingerprinting’ of bacteria, archaea and eucarya. We have applied these libraries of probes and the spectral deconvolution software to correctly identify highly complex mixtures of cells in situ by spectral sorting. This new technology for multispectral taxonomic identification (MTID) will benefit clinical and environmental microbiology as well as biotechnology. This instrumentation can also be used with other types of multispectral probes, such as fluorescently labeled antibodies.
Identification of microorganisms, eucaryotic cells, and viruses by a variety of methods has become an essential diagnostic tool in areas such as healthcare, food and water quality testing, and enzyme discovery (Amann et al., 1992; O'Hara et al., 1993; Vandamme, E. J., 1994; Birnbaum et al., 1994; Vandamme, P., 1996; Relman, 1998; Schrenk et al., 1998). Identification is an integral part of biological taxonomy, or the classification of organisms. Its medical uses include confirming bacterial serotypes for epidemiological studies (Birnbaum et al., 1994) and monitoring of nosocomial infection (Andersen, 1995). Environmental uses include analysis of water, soil and air, as well as bioremediation monitoring (Schrenk et al., 1998) and studies of population ecology and bacterial phylogenetics (Pace et al., 1986; Ward et al., 1992; Amann et al., 1995). In biotechnology, taxonomic identification can be used for biodiversity screening, bioprocess monitoring and genomic analysis (Amann et al., 1992; Hoheisel, 1997; Head et al., 1998). Traditionally, microbiologists performing bacterial identification have relied on cultivation of organisms, despite the realization that most of them (>99%) are not cultivable by standard methods (Amann et al., 1995; Pace, 1997; Head et al., 1998; Hugenholtz et al., 1998b). Many of these culture-based methods rely on chemical analysis of phenotypic characteristics. For example, there are numerous phenotype-based systems for identifying bacterial and archaeal cultures according to their cellular fatty acid ester content (Osterhout et al., 1991), endogenous enzyme activity and/or antibiotic resistance patterns (O'Hara et al., 1993), and antigenic markers (Porter et al., 1993). In the case of antigenic markers, fluorescently labeled antibodies can be used to specifically identify bacterial serotypes, such as the common food pathogen,
E. coli
0157:H7 (Restaino et al., 1997; Seo & Frank, 1999). This technique is useful for accurately identifying microorganisms at the species and subspecies level. Recent advances in combinatorial mutagenesis and phage-display technology have also made it possible to create peptides and proteins that have the affinity and specificity of antibodies but are not derived from antibody molecules per se.
More recently, molecular based methods have been developed to examine he diversity of microorganisms without the need to isolate or culture them. One class of methodology takes advantage of the conserved nature of protein synthesis in all cellular organisms. With about 10,000 partial or complete sequences now available for comparison, the small subunit ribosomal RNA (rRNA) (which contains the 16S rRNA in bacteria and archaea and the 18S rRNA in eucarya) is currently the molecule of choice for identifying organisms at the species level. Other rRNA targets include the large subunit 5S or 23S rRNA (in bacteria and archaea) and the large subunit 5S, 5.8S and 28S rRNA (in eucarya). Molecular strategies based on PCR, cloning, sequencing, and probing have enabled biologists to examine the total microbial community in a sample without any a priori knowledge of the species present in the mixture (Amann et al., 1995). Although rRNA-based identification is only accurate to approximately the level of species, its tremendous versatility makes it extremely valuable for high-throughput screening and identification of microorganisms.
The information gained from 16S/18S rRNA sequence comparisons can be used to deduce detailed phylogenetic relationships based on evolution. An evolutionary distance map generated from 16S rRNA sequence data highlights the major lineages of Bacteria and Archaea (FIG.
1
). The highly conserved portions of 16S/18S rRNA are ideal for designing primers that will amplify 16S/18S rRNA genes from all three domains of life (Bacteria, Archaea, and Eucarya). At the other extreme, primers can be designed to highly variable regions of 16S/18S rRNA and thus amplify only a particular species or genus in a mixture of microorganisms. Likewise, fluorescent DNA hybridization probes based on 16S/18S sequencing information can be constructed to identify organisms in a large group (i.e., phylum) or in a localized group (i.e., genus), depending on whether the probe sequence is complementary to a conserved or variable region of the 16S/18S rRNA, respectively. Ribosomal RNA is a particularly convenient and attractive hybridization target for quantitative microscopy because a typical
E. coli
cell contains approximately 20,000 ribosomes (Neidhardt, 1987), and thus ~20,000 copies of the target sequence. These probes can also be made using polymers other than DNA. Such polymers include RNA as well as nucleic acid analogues, such as peptide-nucleic acids, phosphorothioates, and morpholinos. Probes can be covalently labeled with fluorophores or other spectroscopically identifiable labels to enable in situ hybridization and identification by fluorescence or other spectroscopic imaging microscopy (Amann et al., 1990). The probes can also contain fluorophores designed to be FRET (fluorescence resonance energy transfer) pairs, such as molecular beacons.
PCR has been an extremely powerful tool for analyzing samples and constructing databases of sequences. It has been used to amplify the 16S-rDNA genes from microorganisms isolated from highly diverse and extreme environments, as well as from clinical sources (Hugenholtz et al., 1998b; Relman, 1998). Unknown organisms are being identified at the level of new phyla, expanding on the bacterial line of descent. Many of these new phyla do not have cultured representatives, and yet PCR analysis indicates that they are abundant in the environment. These organisms are completely novel, and they may be a rich source of new antibiotics, enzymes, and other bioactive compounds for medicine and biotechnology (Short, 1997). Recently, attempts have been made to reduce the sequencing load and to increase the screening throughput by employing restriction fragment leng
Bylina Edward
Coleman William
Dilworth Michael R.
Robles Steven J.
Silva Christopher M.
Fenwick & West LLP
Kairos Scientific Inc.
Mariam Daniel
Patel Shefali
Shuster Michael J.
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