Construction and use of catalogued nucleic acid libraries that contain advantageously adjusted representations of defined components

Abstract

A process for constructing a catalogued nucleic acid library in which the proportional representation of the constituents is adjusted to advantage through the use of disclosed technologies for positive and negative selection. The resultant benefit is that significantly fewer library constituents will need to be screened in order to identify a potentially desired constituent. Moreover, library constituents that previously would have been essentially “lost” are now recoverable. Preferred embodiments of this invention include the cataloguing, normalization, and enrichment of library constituents. By way of example, but not limitation, this technology is serviceable for constructing a library that contains an adequate representation of desirable constituents that (1) are initially found in low-copy numbers within a sample source or (2) originate from an organism that is problematic to culture. Applicable uses of this invention include any library-screening endeavor previously hindered by logistical impediments. 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. By disclosing the construction and screening of high yielding nucleic acid libraries from mixed and uncultivated organisms, the instant technology eclipses former boundaries in the area of biological discovery and enables the full breadth of biological diversity to be accessed in the search for previously undiscovered genes and gene products. The benefits of the present invention are seen to extend to areas of diagnosis, medicine, agriculture, manufacturing, and academia.

Description

[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...

AI Technical Summary

Benefits of technology

[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.

[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.

[0054] To “normalize” a sample refers to subjecting the sample to a process such that the proportional representation of each of the components is resultantly made more comparable if not virtually indistinguishable. Accordingly, in one embodiment, “normalization” is exemplified by, but not limited to, reducing the redundancy of clones from overly abundant species, or alternatively increasing the proportional representation of clones from rare or underrepresented species, or both.

[0055] To “selectively enrich” (or “enrich” for short) a sample refers to subjecting the sample to a process such that the proportional representation of at least one component or group of components is resultantly enhanced.

Problems solved by technology

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.

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.

Moreover, (5) the abundance of a desired nucleic acid may be prohibitively low even within a single organism species.

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.

However, previously attempts at screening mixed populations were unfeasible if not impractical and were avoided because of the cumbersome procedures required.

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.

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.

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 cultivation of each organism represented in any given environmental sample would require significant time and effort.

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.

Images