Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-09-23
2002-06-04
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06399334
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the fields of molecular biology and genetics. The invention relates generally to methods for producing normalized nucleic acid libraries, such that the variation in the abundance of the individual nucleic acid molecules in the library is substantially reduced (e.g., to no greater than about two orders of magnitude). The invention also relates to normalized libraries produced by these methods, to nucleic acid molecules isolated from these libraries, to genetic constructs (e.g., vectors) comprising these nucleic acid molecules, and to host cells comprising such normalized libraries.
BACKGROUND OF THE INVENTION
The elucidation of the mechanisms that dictate the normal functioning of living cells requires a detailed understanding of the information encoded in all of the genes (also referred to here synonymously as the genome). To map and sequence the genes contained in the genomes of different organisms, messenger RNA (mRNA) sequences, which are representative of the genes of the genome, are typically used to evaluate the genetic make up of the particular cell or organism of interest. However, the mRNAs (estimated to number 100,000 in human) are produced at different levels within different cell types at different points in development (e.g., there are less than one copy per cell of some mRNAs and there are millions of copies per cell of others). These mRNAs, their developmental and cell-type specific regulated expression, and their translation into protein is what produces the unique character of a particular cell type. For example, adult muscle cells produce high levels of myoglobin mRNA whereas mature red blood cells contain high levels of hemoglobin. In the fetus, hemoglobin is produced by the liver; however, following birth, the type of hemoglobin produced and the tissue source both change, due to changes in gene expression.
An understanding of the molecular details of normal functioning of cells is essential in order to understand and treat inherited diseases where the regulation and expression of one or more genes may have changed. Integral to this goal is the production of libraries of cloned nucleic acids from which all or substantially all of the members of the libraries can be isolated with approximately equal probability.
A normalized library with a lower range of its members relative concentrations, for example as low as about 2-4 fold, would have the advantage of making essentially all of the mRNAs available for isolation and subsequent analysis. This type of library would further the understanding of the normal function of individual genes and the genome in general. However, none of the methods reported heretofore have resulted in the production of normalized nucleic acid libraries where essentially all of the nucleic acid molecules or genes expressed in a particular cell or tissue type are represented and can be isolated with high probability. Although some investigators have attempted to normalize (i.e., reduce the variation in the relative abundance of the components of the population of nucleic acid molecules), none have been successful at bringing the relative abundance of the total population to within a range of two orders of magnitude (Bonaldo, M., Lennon, G., Soares, M. B.,
Genome Res.
6:791-866 (1996); Ko, M. S. H.,
Nucl. Acids Res.
18:5705-5711 (1990); Pantanjali, S. R., et al.,
Proc. Natl. Acad. Sci. USA
88:1943-1947 (1991); Soares, M. B.,
Proc. Natl. Acad. Sci. USA
91:9228-9232 (1994)). The resulting “normalized” libraries have failed to provide the quantity of novel information needed to understand the expression of most genes. Thus, there exists a current need for methods of producing normalized nucleic acid libraries, and for normalized nucleic acid libraries produced by such methods.
BRIEF SUMMARY OF THE INVENTION
The present invention meets this need by providing methods for producing normalized nucleic acid libraries (i.e., libraries of cloned nucleic acid molecules from which each member nucleic acid molecule can be isolated with approximately equivalent probability). In particular, the invention relates to methods for normalization of a nucleic acid library, which may be a single-stranded or double-stranded cDNA library, comprising:
(a) synthesizing one or more nucleic acid molecules complementary to all or a portion of the nucleic acid molecules of the library, wherein the synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules (which may be RNA molecules or DNA molecules);
(b) incubating a nucleic acid library to be normalized with the haptenylated nucleic acid molecules (e.g. also referred to as driver) under conditions favoring the hybridization of the more highly abundant molecules of the library with the haptenylated nucleic acid molecules; and
(c) removing the hybridized molecules, thereby producing a normalized library.
In a preferred aspect of the invention, the relative concentration of all members of the normalized library are within one to two orders of magnitude. In another preferred aspect, the invention allows removal or elimination of contaminating nucleic acid molecule from the normalized library. Such contamination may include vectors within the library which do not contain inserts (e.g. background). In this manner, all or a substantial portion of the normalized library will comprise vectors containing inserted nucleic acid molecules of the library.
The invention also relates to such methods wherein the conditions favoring hybridization of the more highly abundant molecules of the library with the haptenylated molecules are selected from the group consisting of. (a) a COT equal to or greater than 25; (b) a COT equal to or greater than 50; (c) a COT equal to or greater than 100; (d) a COT equal to or greater than 1,000; (e) a COT equal to or greater than 2,000; (f) a COT equal to or greater than 5,000; (g) a COT from about 10 to 10,000; (h) a COT from about 25 to 10,000; (i) a COT from about 50 to 10,000; (j) a COT from about 1,000 to 10,000; (k) a COT from about 5,000 to 10,000; (l) a COT from about 500 to 5,000; (m) a COT from about 100 to 1000; and (n) a COT of less than 10,000.
In a preferred aspect of the invention, a population of mRNA is incubated under conditions sufficient to produce a population of cDNA molecules complementary to all or a portion of said mRNA molecules. Preferable, such a population of cDNA molecules (e.g. single stranded cDNA) is produced by mixing the population of mRNA molecules (template molecules) with one or more polypeptides having reverse transcriptase activity and incubating said mixture under conditions sufficient to produce a population of single stranded cDNA molecules complementary to all or a portion of said mRNA molecules. The single stranded cDNA molecules may then be used as template molecules to make double stranded cDNA molecules by incubating the mixture under appropriate conditions in the presence of one or more DNA polymerases. The resulting population of double-stranded or single-stranded cDNA libraries may be normalized in accordance with the invention. Preferably, such cDNA libraries are inserted into one or more vectors prior to normalization. Alternatively, the cDNA libraries may be normalized prior to insertion within one or more vectors, and after normalization may be cloned into one or more vectors.
In a particularly preferred aspect of the invention, the library to be normalized is contained in (inserted in) one or more vectors, which may be a plasmid, a cosmid, a phagemid and the like. Such vectors preferably comprise one or more promoters which allow the synthesis of at least one RNA molecule from all or a portion of the nucleic acid molecules (preferably cDNA molecules) inserted in the vector. Thus, by use of the promoters, haptenylated RNA molecules complementary to all or a portion of the nucleic acid molecules of the library may be made and used to normalize the library in accordance with the invention. Such synthesized RNA molecules (which have
Jessee Joel
Li Wu-Bo
Nisson Paul E.
Campbell Eggerton A.
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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