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Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined componentsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidConstruction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060088821, Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a divisional of co-pending U.S. patent application Ser. No. 09/089,787, filed Jun. 3, 1198, which is a continuation-in-part of U.S. patent application entitled "Production and Use of Normalized DNA Libraries", Ser. No. 09/034,724, filed Mar. 4, 1998; which is a continuation-in-part of U.S. patent application entitled "Production and Use of Normalized DNA Libraries", Ser. No. 08/665,565, filed Jun. 18, 1996. FIELD OF THE INVENTION [0002] The present invention relates to the production and screening of nucleic acid libraries, and more particularly to the production and screening of nucleic acid libraries from mixed populations of prokaryotes, eukaryotes, and/or other organisms wherein the proportional representations of defined components of the libraries are adjusted to advantage. Preferred advantageous adjustments include the normalization and the selective enrichment of library components. BACKGROUND OF THE INVENTION The General Problem To Be Solved: [0003] In sum, it is often desirable but logistically unfeasible to isolate a nucleic acid from a library because the desired nucleic acid is too severely underrepresented. This problem arises in the screening of a nucleic acid library constructed from a plurality of heterogeneous organism forms, particularly when a desirable source organism form is disproportionately underrepresented. This problem additionally arises when a desired nucleic acid target is a relatively more rare or optionally a more unstable nucleic acid species when compared to even other nucleic acids that are derived from the same organism form. [0004] One currently favored approach is thus to construct and screen libraries derived from a single organism source. However, the isolation of a single organism species from the rich complexity of an environmentally derived sample often requires culturing or other separation approaches to achieve homogeneity, and consequently this approach is frequently problematic and painstaking, if not unfeasible. Specifically, it has become increasingly appreciated that within the often rich complexity of an environmentally derived sample there may exist (1) a desirable source organism that possesses poorly understood culturing requirements and/or responses, (2) a desirable source organism that is problematic to culture, (4) a desirable source organism that is poorly characterized, and hence is not easily separable or distinguishable, and (5) at least two groupings of culturable but not easily separable organisms that possess incompatible culturing requirements and/or dissimilar culturing responses. Moreover, (5) the abundance of a desired nucleic acid may be prohibitively low even within a single organism species. Alternatively, (6) the abundance of a desired nucleic acid may become drastically diminished upon the subjection its source organism to culturing. Potentially still, (7) the screening process employed may require that there be an exaggerated proportional representation of a desired constituent in order for its presence to be positively identified above, e.g., the background signal. On the other hand, (8) it may be desirable to have a means to access a plurality of heterogeneous organism forms in parallel rather than in series. [0005] The common result, nonetheless, is that--due to logistical considerations--a desired target in a library may be so overwhelmingly outnumbered by undesired components--and particularly by redundant undesired components--that it resembles a needle concealed in a forbiddingly large haystack. Accordingly, the size of the library that must be screened to expect a reasonable chance of success becomes essentially unmanageable. Thus, a particularly desired nucleic acid may be prone to virtual "loss" when subjected to conventional library construction processes and hence becomes unrecoverable during the ensuing screening processes. [0006] In consequence, novel methods to overcome these logistical impediments are highly desirable. In particular, the screening of mixed populations of organisms is a desirable option. However, previously attempts at screening mixed populations were unfeasible if not impractical and were avoided because of the cumbersome procedures required. A Specific Example Of The Problem To Be Solved: [0007] A particular embodiment of the problem addressed by the instant invention is exemplified by, but by no means limited to, the following issue encountered in the area concerned with the search for novel microbial enzymes. Specifically, this area is concerned with the increasing demand in the research reagent, diagnostic reagent, and chemical process industries for protein-based catalysts possessing novel capabilities. At present, this need is largely addressed using enzymes purified from a variety of cultivated bacteria or fungi. However, because less than 1% of naturally occurring microbes can be grown in pure culture (Amann, 1995), alternative techniques must be developed to exploit the full breadth of microbial diversity for potentially valuable new products. [0008] Virtually all of the commercial enzymes now in use have come from cultured organisms. Most of these organisms are bacteria or fungi. Amann et al. (Amann, 1995) have estimated the culturability of microorganisms in the environment as follows: TABLE-US-00001 Habitat Culturability (%) Seawater 0.001-0.1 Freshwater 0.25 Mesotrophic lake 0.01-1.0 Unpolluted esturine waters 0.1-3.0 Activated sludge 1.0-15.0 Sediments 0.25 Soil 0.3 [0009] These data were determined from published information regarding the number of cultivated microorganisms derived from the various habitats indicated. [0010] Other studies have also demonstrated that cultivated organisms comprise only a small fraction of the biomass present in the environment. For example, one group of workers recently reported the collection of water and sediment samples from the "Obsidian Pool" in Yellowstone National Park (Barns, 1994) where they found cells hybridizing to archaea-specific probes in 55% of 75 enrichment cultures. Amplification and cloning of 16S rRNA encoding sequences revealed mostly unique sequences with little or no representation of the organisms which had previously been cultured from this pool, suggesting the existence of substantial diversity of archaea with so far unknown morphological, physiological and biochemical features. Another group performed similar studies on the cyanobacterial mat of Octopus Spring in Yellowstone Park and came to the same conclusion, namely, tremendous uncultured diversity exists (Ward, 1990). Giovannoni et al. (1990) and Torsvik et aL (1990a and 1990b) have reported similar results using bacterioplankton collected in the Sargasso Sea and in soil samples, respectively. These results indicate that the exclusive use of cultured organisms in the screening for useful enzymatic or other bioactivities severely limits the sampling of the potential diversity in existence. [0011] The screening of gene libraries from cultured samples has already proven valuable. It has recently been made clear, however, that the use of only cultured organisms for library generation limits access to the diversity of nature. The uncultivated organisms present in the environment, and/or enzymes or other bioactivities derived thereof, may be useful in industrial processes. The cultivation of each organism represented in any given environmental sample would require significant time and effort. It has been estimated that in a rich sample of soil, more than 10,000 different species can be present. It is apparent that attempting to individually cultivate each of these species would be a logistical impracticality. The alternative approach, specifically, to generate and screen a library that contains a raw and unfiltered proportional representation of all the organisms in the soil sample, likewise presents a logistical impediment. Therefore, novel methods of efficiently accessing the diversity present in the environment are highly desirable. SUMMARY OF THE INVENTION [0012] The present invention overcomes the logistical obstacles encountered when existing technologies employing conventional unadjusted nucleic acid libraries are applied to the screening of an environmentally derived library, by disclosing a technology for the construction and use of a nucleic acid library that contains advantageously adjusted proportions of defined components. Specifically, the instant technology provides a means for constructing a library that is catalogued, i.e. it is characterized with respect to contents, and that is preferably further normalized or enriched with respect to defined components. [0013] By expanding previous logistical frontiers this invention allows for a novel generation of previously unattainable molecules--particularly molecules that are "unclonable" from conventional, unadjusted libraries--to now be detected, cloned, manipulated, expressed, studied, and used. The present invention achieves this goal by providing methods to isolate the nucleic acid molecules from a variety of sources, including eukaryotic cells and tissues, isolated organisms, consortia of microorganisms, primary enrichments, and environmental samples to make libraries in which defined components have been advantageously adjusted with respect to their original representation in the sample sources. Libraries thus constructed can be feasibly screened with respect to the molecular structures and/or the corresponding activities of the component molecules. [0014] In one embodiment, the present invention represents a novel, recombinant approach to generate and screen nucleic acid libraries constructed from mixed organism populations of cultivated or, preferably, uncultivated (or "direct environmental") samples. In accordance with the present invention, libraries with normalized representions of genomes or other nucleic acids from organism that can differ vastly in abundance in natural populations can be generated and screened. This "normalization" approach reduces the redundancy of clones from abundant species and increases the proportional representation of clones from rare species. These normalized libraries are endowed with enhanced yield potentials and allow for greater screening efficiency in the search for genes encoding novel biological catalysts. [0015] Alternatively, in another embodiment, libraries generated according to the instant invention contain an enhanced proportional representation of defined nucleic acids--or groups thereof. This is particularly applicable when a desired library constituent is known to correlate with one or more detectable parameters, particularly when these detectable parameters are serviceable for positively and/or negatively selecting library constituents so as to construct an enriched library. [0016] In sum, the screening of a catalogued library, particularly when the library is further normalized or selectively enriched, provides a means for achieving an enhanced yield potential when compared to the screening of an uncatalogued and furthermore unadjusted library. [0017] Accordingly, the screening of mixed populations of organisms has been made a rational approach because of the availability of techniques described herein, whereas previously attempts were unfeasible if not impractical and were avoided because of the cumbersome procedures required. [0018] Thus, in one aspect the instant invention provides a process for forming a catalogued nucleic acid library from an organism sample comprised of a plurality of organism forms, by (a) forming a derived organism sample from an initial organism sample, such that the proportional representations of the constituents in said derived organism sample are adjusted to advantage by performing in any order, and at least one time, at least one step selected from the group consisting of: (i) subjecting a working organism sample to a process of selection, and (ii) recovering a fraction of a working organism sample having at least one desired characteristic; (b) isolating an initial nucleic acid sample from said derived organism sample; and (c) forming a derived nucleic acid library from said initial nucleic acid sample, such that the proportional representations of the constituents in said nucleic acid library are adjusted to advantage by performing in any order, and at least one time, at least one step selected from the group consisting of: (i) subjecting a working nucleic acid sample to a period of selection, (ii) recovering a fraction of a working nucleic acid sample having at least one desired characteristic, and (iii) assembling a working nucleic acid sample into a nucleic acid library. [0019] Thus, in one preferred embodiment of this aspect, the process comprises the step of recovering a fraction of working organism sample having at least one desired characteristic. Continue reading about Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components... Full patent description for Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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