CA2406391C - Normalized nucleic acid libraries and methods of production thereof - Google Patents

Abstract

The present invention relates generally to methods for producing normalized nucleic acid libraries in which each member of the library can be isolated with approximately equivalent probability. In particular, the present methods comprise subtractive hybridization of a nucleic acid library with haptenylated (e.g., biotinylated, avidinated or streptavidinated) nucleic acid molecules that are complementary to one or more of the nucleic acid molecules of the library, such that the variation in the abundances of the individual nucleic acid molecules in the library is reduced.
The invention also relates to production of normalized nucleic acid libraries (particularly cDNA libraries) in which contaminating nucleic acid molecules have been reduced or eliminated, and to normalized nucleic acid libraries produced by such methods.

Description

«'O 99/15702 P'CT/US98/19948 Normalised Nucleic Acid Laihraries axtd Methods of Production 'Thereof FIELD OF Tl-iI: 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 narmalized 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.
BACitGROUND 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 1~ 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. hlowever, tlye; mRNAs (estimated to number 100,000 in human) are produced at differerZt lev~:ls 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 coil-type specific regulated expression, and their translation into protein is what produces the unique character of a 2~ particular cell type- For example, adult muscle cells produce high levels of myoglobin mRNA whereas mature. red blood cells contain high levels of laernoglobin. (n 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 ita gene expression.
An understanding of the molecular details of normal functioning of cells S is essential in order to understand and treat inherited diseases where the regulation and expression of one or more genes rnay 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 exarnpfe as low as about 'Z-4 fold, would have thc:
advantage of making essentially all of ttae naRNAs available; for isolation and subsequent analysis. This type of library would further the understanding of the normal function of individual genes and the genotne 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 thc~ 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., Genonre Res. 6:791-866 (1996); ICo, M.S.1-I., Nucl. Acids Res. 18:5705-5711 (1990); Pantanjali, S.R., et al., Y'roc. Nail. Acad. Sci. USA 88:1943-1947 '25 (1991); Soares, M.B., Proc. Narl..Ac~ad. .Sci. tlS"A 91:9228-9232 (1994)).
The resulting "normalized" libraries have failed to provide the quantity of novel information needed to understand the expressicm of most genes. Thus, there exists a current need for methods of producing normalized nucleic acid libraries, and for normalized nucleic acid lihraries produced by such methods.

BRIEF SUMMARY OF THE IN~EhTION
An object of the present invention is to provide normalized nucleic acid libraries and methods of production thereof. In accordance with an aspect of the present invention, there is provided a method for normalization of a nucleic acid library comprising:
(a) synthesizing one or more nucleic acid molecules complementary to all or a portion of the ilucleic acid molecules of said library, wherein said-synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules;
{b) incubating a nucleic acid library to be normalized with said haptenylated nucleic acid molecules under conditions favoring the hybridization ofthe more highly abundant molecules ofsaid library with the haptenylated nucleic acid molecules; and (c) removing said hybridized molecules, thereby producing a normalized library.
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 oil 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);

-3 a-(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 ofthe 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 aII
members of the normalized kibrary 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 rmcleic 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 ~4'O 99/15702 PCT/i1S98/19948 haptenylated molecules ar-e selected from the group consisting of (a) a COT
equal t.o or greater than 25; (b) a C'OT equal tc~ or greater than S0; {c) a COT
equal to or greater than 100; (d) a C'.OT 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 t:o 10,000; (h) a C;OT from about 25 to 10,000; (i) a COT from about SO to 10,000; (j) a COT from about 1,000 to 10,000; (k) a COT from about 5,000 to 10,000; (!) a COT from about S00 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 ntRNA molecules. Preferable, such a population ofcDNA molecules (e.g. single stranded cDNA) is produced by mixing the population of mRIVA molecules (template molecules) with one or more polypeptides Craving 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 tire 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 ane 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 art least one KNA molecule from al! or a portion of the nucleic acid molecules (preferably cDNA molecules) inserted in the vector. Thus, Ioy use of' the promoters, haptenylated RNA

W'U 99/15702 PCT/US98/t9948 _5_ molecules comlale;mentary t~~ all or- a portion <rf the nucleic acid molecules of the library may be made and used to normalize the library i~: accordance with the invention. Such synthesized RNA molecules (which have b~er~ haptenylated) will be complementary to all or a portion of the vector inserts of the library.
More highly abundant molecules in the Library may them be preferentially removed by hybridizing the haptenylated RNA molecules to the library, thereby producing the normalized library of the invention. Without being limited. the synthesized RNA
molecules are thought to be representative of tlxe library; that is, more highly abundant species in the library result in more highly abundant haptenytated RNA
using the above method. The relative abundance of the molecules within the library, and therefore, within the haptenylated RNA determines the rate ofremoval of particular species of the library; if a particular species abundance is high, such highly abundant species wilt be removed more readily while low abundant species will be removed less readily from the population. hlormalization by this process thus allows one to substantially equalize the level of each species within the library.
In another preferred aspect of the invention, the library to be: normalized need not be inserted in one or more vectors prior to normalization. In such aspect of the invention, the nucleic acid molecules of the library may be used to synthesize haptenylated nucleic; acid molecules using well known techniques.
For example, haptenylated nucleic acid molecules may be synthesized in the presence of one or more DNA polymerases, one or more appropriate primers or probes and one or more nucleotides (the nucleotides and/or primers or probes may be haptenylated). In this manner, haptenylated hNA molecules will be produced and may be used to normalized the Library in ai;cordance with the invention.
Alternatively, one or more promoters may be added to (or ligated to) the library molecules, thereby allowing synthesis of haptenylated R?~A molecules for use to normalize the library in accordance with the invention. For example, adapters containing one or more pronrotc.rs are added to (lilated to) one or more ends of double stranded library molecule~.s (e.g. cI)l~lA lit7rary prepared from a population w0 99/15702 of mRNA molecules). Such prorrroters may then Ine used to prepare haptenylated fZNA rnolec:ules cornl~lementary to all or a portion of the nucleic acid molecules of the library. In accordance with the invention, the library may then be normalized and, if desired, inserted into one or more vectors.
While haptenylated RNA is preferably used to normalize libraries, other haptenylated nucleic acid rnole:cules may be used in accordance with the invention.
For example, haptenylatecl DNA rnay be synthesized from the library and used in accordance with the invention.
Haptens suitable fbr use in the methods of the invention include, but are not limited to, avidin, streptavidin, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, an enzyme-specific substrate, polymyxin B, endotoxin-neutralizing protein (i~NP), FeT'~, a transferrin receptor, an insulin receptor, a cytokine receptor, CD4, spectrin, focirin, ICAM-1, ICAM-2, C3bi, fibrinogen, Factor X, ankyrin, an integrin, vitronectin, fibronectin, collagen, IS laminin, glycophorin, Mac-l, LFA-l, (3-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody, biotin and combinations thereof. A particularly preferred hapten is biotin.
In accordance with the invention, hybridized molecules produced by the above-described methods may be isolated, for example by extraction or by hapten-~?0 ligand interactions. Preferably, extraction methods (e.g. using organic solvents) are used. Isolation by hapten-ligand interactions may be accomplished by incubation of the tuaptenylated molecules with a solid support comprising at least one ligand that binds the hapten. Preferred ligands for use in such isolation methods correspond to the particular lrapten used, and include, but are not limited 25 to, biotin, an antibody, an enzyme, lipopolysaccharide, apotransferrin, ferrotransff:rrin, insulin, <r cytokine, gp120, y-actin, LFA-l, Mac-I, ,glycophorin, laminin, collagen, fibrone:ctin, vitronectin, an integrin, ankyrin, C3bi, fibrinogen, Factor 7C, ICAM-1, ICA~M-2, spectrin, fodrirx, CD4, a cytokine receptor, an insulin receptor, a transferrira receptor, Fe"', polymyxin B" endotoxin-neutralizing protein 30 (ENP), an enzyme-specific :substrate, protein a1, protein G, a cell-surface Fc receptor, an antibody-specific antigen, avidin, streptavidin or combinations thereof. The solid support used in these isolation methods may be nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose;"' agar, starch, nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead or a microtitre plate. Preferred solid supports are magnetic beads, paramagnetic beads and superparamagnetic beads, and particularly preferred are such beads comprising one or more streptavidin or avidin molecules.
In another aspect of the invention, normalized libraries are subjected to further isolation or selection steps which allow removal of unwanted contamination or background. Such contamination or background may include undesirable nucleic acids. For example, when a library to be normalized is constructed in one or more vectors, a low percentage of vector (without insert) may be present in the library. Upon normalization, such low abundance molecules I S (e.g. vector background) may become a more significant constituent as a result of the normalization process. That is, the relative level of such low abundance background may be increased as part of the normalization process.
Removal of such contaminating nucleic acids may be accomplished by incubating a normalized library with one or more haptenylated probes which are specific for the nucleic acid molecules of the library (e.g. target specific probes).
In principal, removal of contaminating sequences can be accomplished by selecting those nucleic acids having the sequence of interest or by eliminating those molecules that do not contain sequences of interest. In accordance with the invention, removal of contaminating nucleic acid molecules may be performed on any normalized library (whether or not the library is constructed in a vector).
Thus, the probes will be designed such that they will not recognize or hybridize to contaminating nucleic acids (as in the preferred embodiment using the oligodA-Notl 3' biotin probe). Upon hybridization of the haptenylated probe with nucleic acid molecules of the library, the haptenyiated probes will bind to and select desired sequences within the normalized library and leave behind contaminating PC'I'/US98/19948 _h_ nucleic acid molecules, resulting in a sc:lecaed n~~r rnalized library. The selected normalized library naay then be isolated. In a preferred aspect, such isolated selected normalized libraries are single-stranded, and may be made double stranded following selection by incubating the single-stranded library under conditions suf~'rcient to render the rmcleic acid molecules double-stranded.
The double stranded molecules may then be transformed into one or more host cells.
Alternatively, the normalized library may be made double stranded using the haptenylated probe or primer (preferably target specific) and then selected by extraction or ligand-hapten interactions. Such selected double stranded molecules may then be transformed into one or more host cells.
In another aspect of the invention, contaminating nucleic acids may be reduced or eliminated by incubating the normalized library in the presence of one or more primers specific; for library sequences (specific for insert-containing clones, e.g. oligodA-,Notl). 'I°his aspect of the invention may comprise incubating the single stranded normalized library with one or more nucleotides (preferably nucleotides which confer rnrclease resistance to the synthesized nucleic acid molecules), and one or more polypeptides having polymerase activity, under conditions sufficient to render the nucleic acid molecules double-stranded.
The resulting double stranded molecules may then be transformed into one or more host cells. Alternatively, resulting double stranded molecules containing nucleotides which confer nuclease resistance rnay be digested with such a nuclease and transformed into one or more host cells.
In yet another aspect, the elimination or removal of contaminating nucleic acid may be accomplished prior to normalization of the library, thereby resulting 2~ in selected normalized library of the invention. In such a method, the:
library to be normalized may be subjected to any of the methods described herein to remove unwanted nucleic acid molecules and then the literary may then be normalized by the process of tl~e invention to provide for the selected normalized libraries of the invention.

V1'O 99/15702 _9_ In accordance with the invention, double stranded nucleic acid molecules are preferably made single str~rnded before hybridization. Thus, the methods of~
the invention rnay further comprise treating the above-described double-stranded nucleic acid molecules of the library under conditions sufficient to render the S nucleic acid molecules single-stranded. Such conditions may comprise degradation of one strand of the double-stranded nucleic acid molecules (preferably using gene II protein and Exonuclease III), or dena.tur~ing the double-stranded nucleic acid molecules using heat, alkali and the like.
The invention also relates to normalized rmcleic acid libraries, selected IO normalized nucleic acid libraries and transformed host cells produced by the above-described methods.
Other preferred embodiments of the laresent invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.
l5 BRIEF DESC'RIfTION OF THP. DRAWINGS
Figure 1 is a schematic of the phagernid that has been used t:o construct a directionally cloned cDNA library.
Figure 2 is a schematic:. diagram ofthe production of normalized phagemid libraries using subtractive hybridization with a biotinylated total library RNA
:'0 driver referred to synonymously as haptenylated nucleic acid molecules.
Figure 3 is a diagram showing how ' biotinylated target specific probes can be used to produce low-background normalized phagemid libraries also referred to herein as selected normalized libraries.
Figure 4 is a diagram showing how a S' biotinylated target specific probe '?5 can be used to reduce background in normalized phagemid libraries also referred to herein as selected normalized libraries.

~1f)-Figure ~ is a diaeran~ sl~owin~ how nuclease resistant nucleotides and a nuclease yield low-background normalized phai;emid libraries also referred to herein as selected normalized libraries.
Figure 6 is a photograph of an ethidium bromide-stained gel of the enrichment of various T(JF~ cDNAs, that are present at considerably different abundances in an unnormalized cDNA library, at different COTs of subtraction in a normalized human fetal brain cDNA library for which two different background elimination methods have been applied.
Figure 7 is a schematic representation of the normalization of a library using adaptors comprising promoters. Following normalization, the library may be cloned into a vector In this method, removal of contaminating vector sequences rnay be unnecessary, since the selection of background sequences can be undertaken prior LO cloning.
DETAILED DISC'.R~PTION OF TI-IE INVENTION
l~ Definitions In t:he description that follows, a rmmber of terms used in recombinant DNA technology are utilized extensively. In order to provide a clearer and consistent understanding ofthe specificatior~r and claims, including the scope to be given such terms, thf; following definitions are provided.
Library. As used herein, the term "library" or "nucleic acid library" means a set of nucleic acid molecules {circular or linear) representative of all or a significant portion of the DNA <;ontent of" an organism {a "genomic library"), or a set of nucleic acid molecules representative of all or a significant portion of the expressed genes {a "cDNA library") in a cell, tissue, organ or orE;anism. Such 2~ libraries may or may not be contained in one or more vectors.
Normalized A.s used herein, the term "normalized" or "normalized library" means a nucleic acid library that has been manipulated, preferably using the methods of the invention, to reduce the relative variation in abundance among WO 99/15702 I'CT/US9$/1994$
member nucleic acid m~llecules in the library tc7 a range of no greater than about ?p-fold, no greater than about 20-fold, no c,=renter tirar~ about 15-fold, no greater than about 10-fold, no greater than about 7-fold, no greater than about 6-fold, no greater than about 5-fold, no greater than about 4-fi>ld, no greater than about 3 S fold or no greater than about 2-fold.
Driver. As used herein, the term "driver" refers to a population ofnucleic acid molecules (preferably RNA) which are complementary to all or a portion of nucleic acid molecules of a library. Such driver preferably comprises one or more haptens and preferably are in molar excess (greater" than 10, preferably greater than 20 fold) compared to the library of interest. In accordance with the invention, the driver is preferably synthesized from the library to be normalized and then the driver is used to normalize that library.
Background. As used herein., background r~~fers to contaminating nucleic acid molecules which rnay be present in a constructed library. Typical contaminating nucleic acid molecules are vectors in which the library has been constructed but which have lost the inserted nucleic acid molecule (by deletion or otherwise) or which do not contain nucleic acid inserts. The target specific probes or primers described herein will not hybridize to contaminating or background sequences.
2p Vector. As used herein, a "vector" is a plasmid, cosmid, phagemid or phage DNA or other DNA molecule which is able to replicate autonomously in a host cell, and which is characterized by ore or ~r small number of restriction endonuclease recognition sites at which such f~NA sequences may be cut in a determinable fashion without loss of an essential bic>logical function of the vector, and into which DNA may be inserted in order to bring about its replication and cloning. The vector rnay further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, include but are not limited to tetracycline resistance or ampicillin resistance.

w0 99/15702 PCT/US98/19948 _1?_ Primer As used herein, "primer" refer: to a single-stranded oligonuclec~tide that is extended by covtilent bonding of nucleotide monomers during amplification or polymerization of a DNA molecule_ Probe. As used herein, "probe" refers to <3 single stranded oligonucleotide S that may be used to hybridize and/or isolate one or more nucleic acid molecules of interest. Such probes may or may not comprise one or more haptens.
Template. The term "template" as used herein refers to a-double-stranded or single-stranded nucleic acid molecules which are to be amplified, synthesized or sequenced. In the case of a double-stranded molecules, denaturation of its strands to form a first and a second strand is preferably performed before these molecules may be amplified, synthesized or seqt.tenced, or the double stranded molecule may be used directly as a template. For single stranded templates, a primer, complementary to a portion of the template is hybridized under appropriate conditions and one or mare polymerises may then synthesize a nucleic acid molecule complementary to al! or a portion of said template.
Alternatively, for double stranded templates, one or more promoters (e.g. promoter) may be used in combination with one or snore polymerises to make nucleic acid molecules complementary to all or a portion oh the template. The newly synthesized molecules, according to the invention, may be equal or shorter in length than the ?0 original template.
Incorporating. 'The term "incorporating" as used herein means becoming a part of a DNA and/or RNA molecule or prirnecr.
Amplification. As used herein "amplification" refers to any in vitro method for increasing the number of copies of a rmcleotide sequence with the use 2~ of a polymerise. Nucleic acid amplification results in the incorporation of nucleotides into a DNA and!or ILNA molecule or primer thereby forming a new molecule complementary to a template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules.
As used herein, one amplification reaction may consist of many rounds of 30 replication. DNA mnplificat.ion reactions ittciudce, for ehample, polymerise chain ii'O 99/15702 PC1'lUS9$1I9948 reactions (PCR) ()ne f(~R reaction noay consist of ~ to 100 "cycles" of denaturation and synthesis of a DNA molecule.
Oligonucleotide. "Oligonucleotide" refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides which are joined S by a phosphodiester bond between the 3' position of the deoxyribose or ribose of one nucleotide and the S' position of the deoxyribose or ribose of the adjacent nucleotide. A blocking oligonucleotide refers to oligonucleotides which are used to prevent hybridization of a nucleic acid molecules (e.g. probe or a primer) to unwanted or undesired molecules. For example, thc: blocking oligonucleotide may prevent the S' and in some cases the ~' end sequences of the driver components from hybridizing to the library vector.
Nucleotide. As used herein "nucleotide" refers to a base-sugar'-phosphate combination. Nucleotides are monomeric units ofa nucleic acid sequence {DNA
and RNA). 'The term nucleotide includes r-ibonucleoside triphosphate ATP, UTP, GTP, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof . Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein also refers to dideoxyribonucleoside '?0 triphosphates (ddN'rPs) and their derivatives. Illustrated examples of dideoxyribonucleosicle triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddT'fP. According to the present invention, a "nucleotide" nuay be unlabeled or cletectably labeled by well known techniques.
Detectable labels include, for example, radioactive isotopes, fluorescent labels, 2~ chemiluminescent labels, bioluminescent labels acrd enzyrne labels.
Hybridization. rl"he terms "hybridization'" and "hybridizing" refers to base pairing oftwo complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double-strarnded molecule. As used herein, two nucleic acid molecules may be hybridized, althoul;h the base pairing is not completely 30 complementary. Accordrngh;, rtrismatctred bases do not prevent hybridization of two nucleic acid melee:ales provided that appropriate conditions, well known in the art, are used Irt the present irrvention, the term "hybridization" refers particularly to hybridization of a driver to the library to be normalized.
Other terms used in the fields of~recombinant DNA technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts.
Overview The present invention is generally directed to methods for producing normalized nucleic: acid libraries, and to normalized libraries produced by these methods. In one preferred embodiment of the irovention, the normalized library produced is a cDNA library, which may be single-stranded or double-stranded.
According to the invention, normalization of a nucleic acid library is accomplished using haptenylated nucleic acid molecules (i.e., nucleic acid molecules having covalently coupled thereto one or more hapten molecules, such as those described below) which will hybridize more rapidly to the more highly abundant nucleic acid molecules of the library. Such haptenylated nucleic acid molecules are referred to as a driver. This hybridization farms complexes of nucleic acid molecules which may then be removed (thereby reducing the abundance of the bound nucleic acid molecules in the library), preferably via Ggand-hapten interactions or by extraction techniques. It has been discovered that, by the methods of the invention, normalized nucleic acid libraries having a maximum variation in abundance of the member nucleic acid molecules no greater than about 2- to about 10-fold may be produced. Moreover, the methods of the invention provide normalized libraries which have significantly reduced background. Thus, the invention provides rnethods for producing nucleic acid libraries, particularly cDNA libraries, from which each member nucleic acid molecule can be isolated with approximately equivalent probability, regardless of its copy number in the original library.

W'O 99115702 PC'r/US98/19948 _ 15_ Sources of Nucleic Acid l,ibr:rries CJsin~; the methods of the invention, normalized nucleic acid libraries, particularly normalized cDNA libraries, may he prepared from a variety of nucleic acid libraries. Such libraries to be normalized may be prepared using standard techniques or rnay be obtained comnuercially (Life "Technologies, lnc., Rockville, MD). Nucleic acid libraries for use in the present invention include those comprising populations of single-stranded or double-stranded nucleic acid molecules, or preferably populations of single-stranded or double-stranded DNA
molecules. iYlore preferred nucleic acid libraries to be normalized in accordance with the invention include chose comprising complementary DNA (cDNA) libraries. Such cDNA libraries (double stranded or single stranded) rrray be made using well known techniques using messenger RNA or polyA+ RNA or may be obtained commercially, for example from Life Technologies, lnc. (Rockville, Maryland), or other coriimercial saurces that will be familiar to one of ordinary skill. cDNA libraries used in accordance with the invention are preferably made with reverse transcriptases having substantially reduced RNase H activity (see below). The pCMVSPORT vectors for libr<3ry construction is preferred and Life Technologies, Inc. (Rockville, MDT) cDNA libraries are housed in these vectors.
In a preferred aspect of the invention, the nucleic acid molecules ofthe library may be contained in one or more vectors, such as plasmids, cosmids or phages.
Ln accordance with the invention, the nucleic acid libraries maybe prepared from populations of nucleic acid molecules obtained from natural sources, such as a variety of cells, tissues, organs or organisms. , Cells that may be used as sources of nucleic acid molecules may be prokaryotic (bacterial cells, including those of species of the genera Issche.richia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcws, C:lostridiuru, C~hlamydia, Neisseria, Treporrema, Myeoplcrsrncr, Bmrelia, L~gicanella, P.seuclcarrrr~rucr,~, Mycohaeterium, ~Flelicobaeter, Erwinia, flgrobacterium, Rhizobium, and flreplornyces) or eukaryotic (including fungi (especially yeasts), plants, protozoans and other parasites., and animals w0 99/15702 PCTlUS98/19948 includins~ insects (ptrrticularly I ~rn.soplrilcr sp;a. cells), nematodes (particularly Caenorhabdilis elegcxrl.s c:ells), arid mammals (particularly human cells)).
Mammalian somatic cells that may 1>e; used as sources of populations or libraries of nucleic acids include blood cells f reticulocytes and leukocytes), endothelial cells, epithelial ells, neuronal cells (from the central or peripheral nervous systems), muscle cells (including myocytes and myoblasts from skeletal, smooth or cardiac muscle), connective tissue cells (including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes and osteoblasts) and other stromal cells (e.g., macrophages, dendritic cells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes) may also be used as sources of nucleic acids or libraries for use in the invention, as may the progenitors, precursors and stem cells that give rise to the above somatic and germ cells. Also suitable for use as nucleic acid sources a.re mammalian tissues c r organs such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin, genitourinary, circulatory, lymphoid, gastrointestinal and connective tissue sources, as well as those derived from a mammalian (including human) embryo or fetus.
Any of the above prokaryotic or eukaryotic cells, tissues and organs may be normal, diseased, transformed, established, progenitors, precursors, fetal or embryonic. Diseased cells may, for example, include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including HIV) or parasites), in genetic or biochemical patholcsgies (e.y., <-ystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or multiple sclerosis) or in cancerous processes.
Transformed or established animal cell lines rnay include, for example, COS
cells, CI-~O cells, VERO cells, BHK cells, I3eL.a cells, I-iepG2 cells, K562 cells, F9 cells and the like. Other cells, cell lines, tissues, organs and organisms suitable as sources of~nucleic acids for use in the present invention will be apparent to one of ordinary skill in the art. 'these cells, tissues, organs and organisms may be obtained from their natural sources, or may be obtained commercially from 17~
sources such as American Type (;ulture (;ollection (Rc~~:kville, Mtaryland) and others that are known to the spilled artisan.
Once the startin~~ i;ells, tissues, organs or other samples arc, obtained, nucleic acid molecules (such as mRNA or poly A t- RICA) may be isolated, and nucleic acid libraries (such as cDNA libraries) prepared therefrom, by methods that are well-known in the art (See, e.g., Maniatis, T., e1 al., Cell 15:687-( 1978); Okayama, 1-l., and Berg, 1'., Mol. Cell. BioL 2:161-170 (1982);
Gubler, U., and Hoftman, B.J., Gene :.'x:263-259 (1983)). As noted above, nucleic acid libraries prepared in such a manner wi(I typically contain a vast range of abundances of member nucleic acid molecules, delyending upon the cell, tissue or organism source, and the stage of development or cell cycle of the source. The methods of the invention may then be used to normalize, or narrow or reduce the relative abundances of nucleic acid molecules in the nucleic acid library.
Production of Normalized Nucleic Acid Libraries In the practice of the invention, nucleic acid libraries are normalized, to produce normalized nucleic acid libraries, by methods that may comprise one or more steps. One preferred method of the invention may comprise, for example:
(a) synthesizing one or more nucleic acid molecules complementary to all or a portion of the nucleic acid molecule, of the library, wherein the synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules (e.g. driver);
(b) incubating a nucleic acid library to be normalized with the haptenylated nucleic acid molecules 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.
According to the invention, haptenylated nucleic acid molecules complementary to all or a portion of the trucleic acid molecules of the library may -1 s-be produced, for example, by incubating the nucleic acid molecules of the library with at least one polypeptide having nucleic acid polymer ase activity and with at least one nucleotide comprising at least one hapten. If one or more primers are used for synthesis, the primers may comprise one or more haptens to produce the haptenylated nucleic acid molecules (without or with the use of haptenylated~
nucleotides during synthesis). Preferred polypeptides having nucleic acid polymerase activity for use in this aspect of the invention include those having reverse transcriptase activity and those having DNA polymerase or RNA
polymerase activity.
Preferred polypeptides having reverse transcriptase activity (i.e., those polypeptides able to catalyze the synthesis of a DNA molecule from an RNA
template) include, but are not limited to, Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMU) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase; Myeloblastosis Associated Virus (MAV) reverse transcriptase, Human Immunodeficiency Virus {HIV) reverse transcriptase, retrovirai reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B
reverse transcriptase, cauliflower mosaic virus reverse transcriptase and bacterial reverse transcriptase. Particularly preferred are those polypeptides having reverse transcriptase activity that are also substantially reduced in RNase H activity (i. e., "RNASE H-" polypeptides). By a polypeptide that is "substantially reduced in RNASE H activity" is meant that the polypeptide has less than about 20%, more preferably less than about 15%, 10% or 5%, and most preferably less than about 2%, of the RNASE H activity of a wildtype or RNASE H+ enzyme such as wildtype M-MLV reverse transcriptase. The RNASE H activity may be determined by a variety of assays, such as those described, for example, in U.
S.
Patent No. 5,244,797, in Kotewicz, M.L., et al., Narcl. AcidsRes. !6:265 (1988) and in Gerard, G.F., et al., FOCUS 14(5):91 (1992).
Suitable RNASE H' polypeptides for use in the present invention include, but are not limited to, M-_ic~_ MLV I-I- reverse transcriptase, IZSV I-l reverse transc-riptase, AMV 1I-reverse transcriptase, RAV H reverse transcriptase, MA V H reverse transcriptase, HIV
H- reverse transcriptase, anti Sut~F,itSct~tt~i'T"~ I reverse transcriptase and SUYEjZSeRII~'T~T"" II reverse transcriptase which arse available commercially, for example from Life Technologies, lnc. (Rockville, Maryland).
Other polypeptides having nucleic acid polyrnerase activity suitable for use in the present methods include; thermophilic DNA polymerises such as DNA
polymerise I, DNA polymerise ILl, Klenow fragment, T7 polymerise, and TS
polymerise, and thermostable DNA polymerises including, but not limited to, 7lrermus Ihernrophilrr.r ('Ilh) DNA polymerise, l~rermrrs crquaticus (Taq) DNA
polymerise, Thermotoga neopolitana (7rro) DNA polymerise, Thernrotoga rnaritima (Tnra) DNA poiyrnerase, 7lrermococc:rcs li~orali.s (Tli or VENT~) DNA
polymerise, Pyrococcus fi~rios~us (I'fiz or I)EEPVENT~) DNA polymerise, Pyrococcu.r woosii (f'wo) DNA polyrnerase, l3ac°illu.r sterothernrophilus (Bst) DNA polyrnerase, .Sulfolobus acidocaldarius (Sac) DNA polymerise, Thermoplasrrrcr acidophilrrrrr ('Icrc) DNA polymerise, Thermus flavu.s (TfllTub) DNA polymerise, Thermu.s rubes (7i-u) DNA polymerise, Thermus brockianus (DYNAZYNN1~~) DNA polymerise, Nletharrobaeterium thermoautotrophicum (Mth) DNA polymerise, and mutants, variants and derivatives thereof.
RNA polymerises preferably used in the invention may include SP6 RNA
polymerise, T7 RNA polymerise, T3 RNA polymerise and the like. With the use of RNA polymerises, one or more promoters (e.g. SI'6 promoter, T i' promoter, etc.) are typically used. For example, double stranded DNA molecules (or double stranded library) containing orre or more promoters are used in combination with ?5 one or more ILNA polymerises to make haptenylated RNA molecules complementary to all or' a portion of the double stranded library template.
Preferably, such RNA molecules arc in large molar excess compared to the templates. Ln accordance with tln~ invention, such promoters may be provided by the vector in which the library molecules are cloned or by adapter molecules (e.g.
double stranded oligonucleotides) which are added to the library molecules.
When using such adapter molecules, the adapters ( which prefer ably comprise one or more promoters) ar-a added to the library rn«lec:ules Preferably, the library molecules are double stranded linear molecules (e.g double stranded linear cDNA
produced after first and second synthesis), arid the adapters may be added using standard techniques (e.g. ligases) to one or both termini of such molecules.
Preferred nucleotides for use in the methods of the present invention include, but are not limited to, ribonucleosidc: triplrosphates such as ATP, UTP, CTP, GTP and derivatives thereof; and deoxyribanucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, crr derivatives thereof. Such derivatives include [cxS~dATf, 7-deaza-dGTI' and 7-deaza-dAT'P, or the corresponding ribonucleoside triphosphates in which deoxyribose has been replaced by ribose. According to the invention, the nucleotides or derivatives thereof preferably comlorise one or more hapterr molecules covalently bound thereto.
Preferred hapten molecules for use in these methods include, without limitation: (i) biotin; (ii) an antibody; (iii) an errzynre; (iv) lipopolysaccharide; (v) apotransferrin; (vi) ferrotransferrin; (vii) insulin; (viii) cyokines (growth factors, interleukins or colony-stimulating factors); (ix) gpI20; (x) [i-actin; (xi) LFA-1;
(xii) Mac-1; (xiii) glycophorin; (xiv) laminin; (xv) collagen; (xvi) fibronectin; (xvii) vitronectin; (xviii) integrins oc~(j, and o~,[3~; (xix) irctegrins oc3(3,, oc4(3,, oca(3~, a5y, cx~(3r, a.~[i3, 0~,[33and a"[36; (xx) integrins a,[3,, a.zj3,, a3[3, and oc"[33; (xxi) integrins a.,(3,, a,2(~,, oc3(3,, ocb[3,, a,~3r and cx~,[35; (xxii) a.nkyrin; (xxiii) C3bi, fibrinogen or Factor X; (xxiv) ICANI-1 or ICAM-2; (xxv) spectrin or fodrin; (xxvi) C',D4;
(xxvii) a cytokine (e.g., growth fa<aor, interleukin or colony-stimulating factor) receptor;
(xxviii) an insulin receptor; (xxix) a transferrin receptor; (xxx) Fe*";
(xxxi) polymyxin B or endotoxin-neutralizing protein (FNP); (xxxii) an enzyme-specific substrate; (xxxiii) protein r~, protein G, a cell-surface Fc receptor or an antibody-specific antigen; (xxxiv) avidin and streptavidin; and combinations thereof. A
particularly preferred hapten for use in the methods of the invention is biotin. 'fhe s0 haptenylatecl nucleic acid molecules, in which one° or more hapten molecules are attached (preferalsly covalently) to one or n~,sre nucleotides of the nucleic acid molecule, may be pr oducecl using conventiolral csrganic sent nesis methods that will be familiar to one of ordinary skill in the art. I-Tor example, the nucleic acid molecule may be biotinylated at. the S' terminus by krrst producing 5' amino (NHZ) groups followed by C:ab-NHS ester' addition (Langer, P.R., et al., Proc. Natl.
Acac~ Sci. LIS:9 7~:fi633 (1981)). In a particularly preferred aspect of the invention, a haptenylated nucleic acid molecule, which may be an RNA molecule or a DNA molecules, comprising one or more, two or more, three or snore or four or more hapten molecules, most preferably biotin molecules, is prepared.
Once the haptenylated nucleic acid molecules that are complementary to the nucleic acid molecules of the library havf: been produced, they are used to normalize the nucleic acid library by hybrrdlzatron. Specifrcally, the nucleic acid library to be normalized is lorefer<rbly incubated with a molar excess of the population of haptenylated rnrcleic acid molecules (e.g. greater than or equal to 10 fold or preferably greater than or equal tc> 20 fold molar excess), prepared as described above, under conditions favoring the more rapid hybridization of the haptenylated nucleic acid molecules to thc: more highly abundant nucleic acid molecules and less rapid hybrid to the less abundant nucleic acid molecules present in the library. Such conditions favoring hybridization may comprise, for example, incubating the library to bc; normalized with tine haptenylated nucleic acid molecules at a range of COTS. C'O'T is the product of the starting concentration of nucleic acid (moles of nucleotide per liter , ~'.o) =rnd time (seconds, t).
The COT
is obtained by converting the concentration of rt:acting nucleotides and time of hybridization into standard units (rnol~sec~l,,-' or M~sec). As described in detail 2~ in the Examples below, particularly preferred (~O~I's far use in the present methods include, but are not limited to: a C.'.O'i~ equal to or greater than 25; a COT
equal to or greater than 50; a CO's equal to or greater than 100; a COT equal to or greater than 200; a (.'O'T equal to or greater than 250; a COT equal to or greater than 500; a COT equal to or greater than I U00; and a C0T of less than about 10,00(). Alternatively, hybridization conditions consisting of a range of COTs may be used, including a COT from about 10 to about 10,000: a COT from about 25 to about 10,000; a COT from about 50 to about 10,000; a COT from about 100 to about 10,000; a COT from about 200 to about 10,000; a COT from about 2S0 to about 10,000; and a COT from about 500 to about 10,000. Other hybridization S conditions suitable for use with the present methods will be apparent to one of ordinary skill and may be determined with only routine experimentation.
Under these conditions, the haptenylated nucleic acid molecules hybridize more rapidly to the more highly abundant nucleic acid molecules present in the library and less rapidly to the less abundant members. The hybridization complexes formed between the library and the haptenylated nucleic acid molecules may then be removed by a variety of methods, resulting in the reduction in copy number of the highly abundant nucleic acid molecules in the library and thus producing a normalized nucleic acid library.
According to the invention, removal of the complexes is accomplished by ligand-hapten interactions using a ligand which binds specifically to the hapten that is bound to the haptenylated nucleic acid molecules. In a preferred such method, the ligand may be bound, preferably covalently, to a solid support such as nitrocellulose, diazocellulose, glass, polystyrene (including microtitre plates), polyvinylchloride, polypropylene, polyethylene, dextran, SepharoseMagar, starch, nylon, or beads, which may be latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads or glass beads. Particularly preferred solid supports are magnetic beads, paramagnetic beads and superparamagnetic beads, which are commercially available, for example from Life Technologies, Inc. (Rockville, MD), Dynal A.S. (Oslo, Norway), or from Sigma (St. Louis, Missouri).
2~ Coupled to these solid supports may be any ligand capable of binding the hapten used to haptenylate the nucleic acid molecules. Examples of suitable ligands for use in the present methods (which correspond in order to the hapten molecules listed above) include without limitation: (i) avidin and streptavidin; (ii) protein A, protein G, a cell-surface Fc receptor or an antibody- specific antigen;
(iii) an enzyme-specific substrate; (iv) polymyxin B or endotoxin-neutralizing W'O 99/15702 PC'I'ltlS98/19948 73_ protein (ENI'); (v) f~e'-"; (vi) a tran,~ferrin recr~ytor: (vii) au insulin receptor; (viii) a cytokine (e.,~=. , growth factor. mte~-leukin or colony-stimuiaiing factor) receptor;
{ix) CD4; (x) spectrin or fodrin; {xi) 1CAM-I or I(::AIv1-2; (xii) C3bi, fibrinogen or Factor X; (xiii) ankyrin; (xiv) integrins cxr(3,, a2~3,, a,(3,, a~(3,, a7(3, and a~(35 ;
(xv) integrins a,~3,, a2(3,, a;~, and a,,~3~; (xvi;) integrins oc3(3,, a,(3,, a4/~7, as(3,, a"~,, 0L~~3, a~,~3 and a"ø~,; (XVI1) integrins a"~r and OC~,~3; (XVrl1) vitronectin;
(xix) fibronectin; (xx) collagen; (xxi) laminin; (xxii) glycophorin; (xxiii) Mac-l;
(xxiv) LFA-1; (xxv) (3-actin; 4 xxvi) gp 120; (xxvii) cytokines (growth factors, interleukins or colony-stimulating factors); (xxviii) insulin; (xxix) ferrotransferrin;
1 fl (xxx) apotransferrin; {xxxi) lihopolysaccharide; (xxxii) an enzyme;
(xxxiii) an antibody;(xxxiv) biotin; and combinations thereof. Preferred ligands include avidin and streptavidin. Of course, the choice of ligand will depend upon the choice of hapten used in the production of the haptenylated nucleic acid molecule;
appropriate ligands for use in the methods of the invention will thus be apparent to one of ordinary skill in the art. Linkage of the ligand molecules) to the solid support can be accomplished by airy method of ligand coupling such as covalent, hydrophobic or ionic coupling (including coating) that will be familiar to one of ordinary skill in the art. For example, in a preferred aspect of the invention where the haptenylated nucleic acid molecules comprise biotin. a biotin-binding ligand such as avidin or streptaviclin rnay bc; linked to the solid support. In a particularly preferred such aspect, the solid support used is avidin- or streptavidin-coupled magnetic, paramagnetic or superparamagnetic beads.
Typically, conditions favoring ligand-Itapten interactions include incubation in a buffered salt solution, preferably a TINS-, phosphate- HEPES- or carbonate-buffered sodium chloride: solution, more preferably a TR1S-buffered sodium chloride solution, still more preferably a sc~lntiora comprising about 10-100 mM
TRIS-I-ICl and shout 300-2000 mlVt NaC'1, arid most preferably a solution comprising about 10 mM TR1S-HCl and about 1 M IsaCl, at a pH of about 6-9, more preferably a pI~ of abo,~t 7-F~, still mcsre preferably a pH of about 7.2-7.6, and most preferably a pl-1 ofa~:~out 7.5 Incubation is preferably conducted at 0°C' to about 25 °C, and most ~ ~reterably at about :? 5 "c. ~, fbr about 30-120 minutes, preferably about 45-9(.i rr~inutes, and most pref'erat>ly about G() minutes, to allow the binding of the haptenylated nucleic acid molecules (and thus the complementary library nucleic acid molecules to which they are hybridized) to the ligand-coupled solid support.
Once the haptenylated complexes have been bound to the solid phase support, the normalized nucleic acid library, comprising nucleic acid molecules of a lower range of abundances than the input library, may be collected from the supernatants or eluates (i.e., the unbound materials in solution). For example, in a preferred aspect in which biotinylated nucleic acid molecules are bound to avidin or streptavidin; or a avidin- or strept<rvidin-coupled solid phase, the nucleic acid molecules comprising the normalized nucleic acid library, such as a normalized cDNA library, may be obtained by gently aspirating and collecting the supernatants. In a particularly preferred aspect in which avidin- or streptavidin-coupled magnetic, paramagnetic, or superparamagnetic beads are used as the solid support, the biotinylated nucleic acid-containing beads may be segregated from the supernatants using a magnet (such as a Magna-Sep Magnetic Particle Separator;
Life Technologies, Inc.) and the supernatants rnay be withdrawn using a pipette.
Removal of the haptenylated complexes is preferably accomplished by extraction 2U with an organic solvent (e.g. phenol, chloroform etc.). The above described approaches result in the production of a norrualized nucleic acid library, which may be single-stranded or double-stranded and which may be used immediately, stored until use, or processed and further purified in accordance with the invention of by technidues that are well-known in the literature (see, e.~,~., Gubler, U., and Ho~man, 8..1., Gene 25:263-269 (1!)83); Krug, I~1.S., and Berger, S.L., Meth.
E~TZymol. 152:316-325 (1987); Sambrook, J., er al., Molecular C_.'loning_ A
Laboratory Mayrual, 2nd ed., C:old Spring I-larbor, NY: Cold Spring Harbor Laboratory Press, pp. 8.60-8.63 (1987)), and others that will be familiar to one of ordinary skill in the art.

CVO 99/15702 PCT/US98/1994g _p 5_ E3ackground Heduc.tion or Llimination The invention also prrwides metlrocis far the production of a selected normalized nucleic acid library with very low nor-recombinant and rearranged clone background. As used herein, a selected normalized library is~ a library in S which one or more specific nucleic acid molecules or sets of nucleic acid molecules have been enriched in the normalized library and other nucleic acid molecules of less interest have been removed by one or several approaches described herein. Thus, the: invention further relates to removal of contaminating or background nucleic acid molecules from the normalized library. In accordance with the invention, such removal or elimination of contaminating nucleic acids may be performed prior to or after normalization. Typical contaminating nucleic acid molecules in a library are vector molecules v~,'hiclr do not contain nucleic acid molecules of the library (where. the vector failed to receive an insert or the vector lost the insert by deletion during propagation of tl7e source library).
In accordance with the invention, target-specifrc probes (e.g. oligodA-;VotI) may be used in a number of methods to reduce or remove contaminated nucleic acids from the librsiry of interest. Such prcabes are target-specific in that they recognize and hybridize to molecules of the library molecules but not to contaminating nucleic acid sequences (such as vectcors without library inserts. One such means involves usin; one or more hahterrylated target-specific probes to capture or isolate the library of interest. In suc;h ~rsethods, the normalized library is preferably single-stranded (or, if double-stranded, is made single-stranded by methods described herein). liy hybridizing the haptenylated prates to the normalized library, the hybridized normalized library may be selected away from contaminating nucleic acid using, for e;xarnple, hapten/ligand interactions or extraction. The resulting single-stranded selected normalized library may then be made double stranded by incubating the library with one or more polypeptides having polymerase activity under conditions sufficient to synthesize double-stranded selected normalized library.

PCTIt1S98/19948 Alternatively, the r~ornialized library i~; hyhridized to a target-specific, haptenylated primer and tire molecules may then be made double-stranded by incubating them with one. or mc3re polypeptidfa having polymerase activity under conditions sufficient to synthesize double-stranded normalized library. In making _'> such molecules double-stranded, one or more nuclease-resistant nucleotides may be used. The double-stranded molecules may then be selected away from the contaminating nucleic acid molecules using, far example, hapten/ligand rnteractrons or extraction.
In both cases, the resulting double stranded selected normalized library of the invention may then be transformed into one c>r more host cells in a further selection step. In accordance with the invention, single stranded molecules are transformed at a very low fi-eduerrcy while double stranded molecules are transformed at a very high frequency. Thus, transformation allows for an additional selection step in which single stranded contaminating molecules are eliminated or removed. For example, when a target specific probe or primer is used in the double stranded synthesis step, port-specific nucleic acids are not primed and thus are not made double stranded and will not be present in the selected normalized library.
In another aspect of the invention, single~stranded selected normalized library selected with the haptenylated probes arc: made double-stranded with primers (preferably target specific primers) and one or more nucleotides which confer nuclease resistance to the synthesized double-stranded molecule.
Digestion with such a nuclease allows removal ofsingle-stranded molecules which have not been made double stranded by the primers. Such double-stranded molecules may then be transformed into one or more host cells as an additional selection step.
In yet another aspect, the selected normalised library rnay be prepared by incubating the sin JTIe-stranded normalized library with one or more target-specific primers which arc riot haptenylated in c;ornbir7ation with one or more nucleotides which confer nuclease resistance Digestion of the mixture provides for the selection of the desirer:l nucleic acid molecrrles anti as a additional selection step, WO 99/15702 PCT'/US98/19948 the resulting double-stranded ms.>lec:ules rnay f,~ transfcrrr~~ed into one or more Host cells.
In accordance with the invention, sinrgle stranded molecules may be made from double stranded by treating double-stranded molecules under conditions S sufficient to render them single-stranded. Such conditions may comprise, for example, deb=,radation of one strand of the double-stranded nucleic acid molecules in the library, such as by using an endonuclease, an exonuclease, and the like, and preferably by using gene Il protein and exonuclease Ill (available from Life Technologies, Inc., Itockville, IvID). Alternatively, such conditions may comprise denaturing the double-stranded molecules with heat, ionic conditions, pH (e.g.
base) and the (ike.
Nucleotides which coni'er nuclease resistance used in accordance with the invention are preferably nucleotide analogs. Such nucleotide analogs include but are not limited to rnethylated nucleotides such ars 5-methyldeoxycytosine, 3-methyldeoxyadenosine, 7-methylguanine and the like. Other nucleotide analogs that inhibit or block exonucleases or reatrictic:~n enclonucleases (nucleases) will be recognized by those skilled in the art. Combinations of nucleotide analojs and suitable enzymes that may be used according to the invention also known in the art (see Life Technologies 1997-1998 C:ataiog and Reference Guide, Chapter 6).
Kits 'the present invention also provides kits for use in production and isolation of normalized and selected normalized libraries. Kits according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more containers, such '?5 as vials, tubes, ampules, bor.tles and the like. 'fhe kit of the invention may comprise the driver for normalizing a library or the components needed to make the driver used to normalize a library (for example, one or more polymerases, one or more adapters comprising promoters, one or more vectors. comprising promoters, one or more haptenyiated nucleotides andlor one or more WO 99/15702 I'CT/US98/19948 haptenylated primers or probes). Such hlt5 Illay ~On7prl~e 0170 l)r ItlOre target specific probes or primc~r5 (whic::h rare haht:c.nylrrted or not). In additional aspects, the kits of the invention may comprise one or more nucleotides (e.g., nucleotides which confer nuclease resistance and/or one or more endonucleases, exonucleases or restriction enzymes, such as gene II protein or exonuclease III
or HhaI, used for digestion of the nucleic acid molecules.
Additional kits provided by the invention comprise one or more containers containing one or more of the above-described normalized nucleic acid libraries or selected normalized nucleic acid libraries of the invention.
The i0 libraries in these kits of the invention may be single-stranded or double-srranded, and are preferably cI)NA libraries.
The kits encompassed by this aspect of the present invention may further comprise one or more additional reagertts (c~.g., suitable buffers) and compounds necessary for using the normalized libraries and selected normalized libraries of the invention.
Uses The present invention oar be used in a variety of applications requiring rapid production and isolation of normalized and selected normalized nucleic acid libraries, particularly cDNA libraries. l~he primary use for such libraries is for 2C~ aene discovery and for preparing gene databases. L.,ibraries prepared by the methods of the invention may be used as sources of template nucleic acid molecules for amplification reactions (such as via P(~R), to rapidly identify and/or clone low copy number nucleic; acid molecules, and to produce polypeptides by =enetic engineering techniques.
The invention tltus is also directed to methods for the amplification of a nucleic acid molecule, and to nucleic acid molecules amplified by to these methods. According to this aspect ofthe invc;ntion, a nucleic acid molecule may be amplified (i.~., additional copies of the nucleic acid molecule prepared) by antpllfylng a nucleic arid molecule (e.~~., a cDNA molecule) contained in a normalized library or selected normalized library of the invention according to any amplification method that is known in the art. Particularly preferred amplification methods according to this aspect of the invention include PCR (U.S. Patent Nos.
4,683,195 and 4,683,202), Strand Displacement Amplification (SDA; U.S.
Patent No. 5,455,166; EP 0 684 315), and Nucleic Acid Sequence-Based.
Amplification (NASBA; U.S. Patent No. 5,409,818; EP 0 329 822).
Most preferred are those methods comprising one or more PCR amplifications.
The invention is also directed to methods that may be used to prepare vectors which comprise the normalized or selected normalized libraries of the present invention, to host cells which comprise these vectors, to methods for the production of a recombinant polypeptide using these vectors and host cells, and to recombinant polypeptides produced using these methods. According to this aspect of the invention, a recombinant polypeptide may be produced by culturing any of the above recombinant host cells under conditions favoring production of a polypeptide therefrom, and isolation of the polypeptide. Methods for culturing recombinant host cells, and for production and isolation of polypeptides therefrom, are well-known to one of ordinary skill in the art.
Vectors are produced according to the invention by inserting, using methods that are well-known in the art, one or more of the nucleic acid molecules of interest into a vector. The vector used in this aspect of the invention may be, for example, a plasmid, a cosmid or a phage. Preferred are vectors comprising cis-acting control regions to the nucleic acid encoding the polypeptide of interest.
2~ Appropriate trar~s-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
In certain preferred embodiments, the vectors are expression vectors that provide for specific expression of the nucleic acid molecules contained in the normalized libraries or selected normalized libraries of the invention, which vectors may be inducible and%or cell type-specific Yarticuiarly preferred among such vectors are those inducible loy e~wircor~mental factors that are easy to manipulate, such as temperature and nutrient additives.
Expression vectors useful in the present invention include chromosomal-, episotnal- and virus-derived vectors, e.g., vc~,c;cors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids, and will preferably include at least one selectable marker such as a tetracycline or ampicillin resistance gene for culturing in a bacterial host cell. Prior to insertion into such an expression vector, the nucleic acid molecules contained in the libraries of the invention may be operatively linked to an appropriate promoter, such as the phage lambda I'L promoter, the E. coli lac, tr p and tae promoters. Other suitable promoters will be known to the skilled artisan.
Among vectors preferred for use in the present invention include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, I'hagescript vectors, Bluescript vectors, pNHBA, pNHltia, pNi~l8A, pNH46A, available from Stratagene;
pcDNA3 available froth lnvitrogen; pGlh, pTr-~;fus, pTrc99a, pE'T-5, pET-9, pKK223-3, pICK233-3, pDR540, pRITS availablefTOmPharmacia; andpSPORTI, pSPORT2, pCMVSPORT 2.0 and pSV~SPORTl, available from Life Technologies, Inc. Other suitable vectors will be readily apparent to the skilled a'.0 artisan.
Representative bast cells that may be used according to the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Preferred bacterial host cells include Eschertchia spp. cells (particularly E. eoli cells and most particularly E. coli strains DH l OB and Stbl2), Bacillus spp.
cells (particularly B. .sarbtilis and B. megatcr~irrm cells), Sn-eptomyces spp. cells, Erwinia spp. cells, h'lebsiella spp. cells and Salmonella spp. cells (particularly S. typhirnur-itrm cells). 1'reterred animal host cells include insect cells (most particularly SpodoPtera_fi ugiher-cia Std and Si21 cells and Ti-ichoplusa High-Five cells) and mammalian cells (,most particularly C',IIO, COS, VERO, BHK and human cells). These and other suitable host cells are available commercially, for w0 99/15702 PCT/U598/19948 _31_ e~;ample from Life 'l~r:chnoloi~i~a, lnc (Roc.kvill:~, Maryland), American 'type Culture Collection (Roc:kville, l,~larylarrd) anti Invi~rogen ( pan Diego, California).
It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and rnay be made without departing fi-orn the scope of the invention or any embodiment thereof. I-Iaving now described the present invention in detail, the same will he more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
Examples ~iample 1. 1'rorluction of Norr~ralize~l cI)NA Libraries from Directionall~~-clo~recl cDNA Libraries The process of constnrcting a normalized cDNA library in the pCMVSPORT 2.0 vector is described in this example (Figures 1 and 2). It consists of i) isolating phagemid DNA from a directionally cloned cDNA
library, ii) converting the double-stranded (ds) circular cDNA library DNA into a) a linear ds template for IRNA polymerase production of biotinylated RNA driver and b) single-stranded (ss) circular DNA using CJeneII and Exonuclease III, iii) combining the driver and ss circular library DNA with two blocking :ZO oligonucleotides in a subtraction Hybridization, iv) repairing the non-subtracted ss circular DNA and v) transforming it into 1. cx~li cells thus producing a primary normalized cDNA library.
Production of circular ss DNA frcam circular ds cDNA library DNA is done in the following way. Digest 10 yg of circular ds cDNA in 1 X CieneIl buffer 20 mIV 'fris~1-ICl (pN-=8), 80 mM NaC'.1, 25 nrM MgClz, 2 mM ~i-mercaptoethanol.
~% glycerol, 5 mg/ml I3SA with 8 1.r1 <JeneII at 3t)"C for =~0 ruin in a final volume of2.00 yl 'herminate the tea<aion by incuh,rtion ~~t 65°C' for 5 min.
.Add 12 ttl of WO 99115702 I'CT/US98/19948 _ j2.
E:xonuclease III, and incubate at 37"C for Bt> min. Add s _-~! (10 LI/yl) ofNntl and incubate the mixture for 1 It at _37"C'. Acid ~ yl of exonu~lease III, and continue to incubate for 1 hour at _37°('. l::xtract twice with phenol/chloroform/isoamyl alcohol (2:24: I) and ethanol I>recipitate. Resuspend the circular ss eDNA in 10 yl of RNASE-free TE. Fetal Brain cDNA library (Life Technologies, Inc., Rockville, MD) was made single stranded by this procedure.
Production of linearized ds cUNA trom c::ircular ds cDNA is as follows.
Digest 50 ~tg of circular ds cDNA with 200 units of Notl (LTI) in 300 ~tl of reaction buffer [5 mM Tris~HC'.l, pH 8.0; I mM MgClz; 10 mM NaCI] for 3 hours at 37~C. Add I00 units of NotI, and incubate an additional 3 hours at 37~C.
Extract twice with phenolichlorofortn/isoamyl alcohol (2:24:1 v/v} and ethanol precipitate. Resuspend the linearized ds cDNA in 30 ~tl of RNA SE-free TE
buffer. Human Fetal Brain cDNA library (Life Technologies, Inc., Rockville, MD) was linearized in this manner.
Production of biotinylated'RNA driver from circular ds cDNA library DNA is done in the following way. Prepare a mi~.-ture of the following components. I .214 ml DEPC-treated water, 400 lrl SX transcription buffer [200 tnM Tris-HCl (pH 7.9), 30 mM MgCl2, 10 rnM spermidine-(HCl)3], 200 ltl rNTP
mix (10 ~.M each ATI', G'fP and lJ'I"P, S l.th%( C'.TI', 20 plt~1 biotin-I4-CTP), 16 pl (20 pg) linearized ds from a Ht.rman Fetal Brain cDNA library (see above), 100 pl 0.1 M DTT, and 70 yl SP6 RNA polymerise (3 S0 unitsiul). The Human Fetal Brain cDNA library (l..ife Technologies, lnc., Rockville, MD) was constructed in pCMV~SI'URT vector which contains a ~'."MV promoter, an SPG and T7 polymerise promoter flankinf°, tho multiple ulonirrg site (MSC) for 1RNA driver synthesis. Mix and incubate at 37"C for 13 hours. Add 1 nil of 7.S M ammonium acetate and 8 ml of ethanol. tool on dry ice far 30 min. microcentrifuge for min at 4°C and resuspend the pellet in 1 ml of'I"I. Heat the solution at GS°C and reprecipitate again. Wash the pellet in 70'% ethane>l, dry and resuspend in 1.92 ml water, 40 Itl of 1 M Tris-HCI, [pI-17.5), heat at b5"C for resuspension. Add 20 pl 1 M MgC:l2, 2U p.l DNasel (2,660 units) tc7 the: resuspended RNA and incubate at 37°C for 1 hr. Transfer the treated RNA to a fresh tube and add 40 ul of 0.5 M
EDTA, incubate at 65°C for 10 min. and precipitate it with I ml of 7.5 M
ammonium acetate plus 8 ml ethanol. Resuspend the pellet in 300 ~I of TE, heat TM
at 65°C to aid resuspension and load onto a 1 cm x 18 cm column (Sephadex G-~0) and collect the first peak detected by UV absorbance at 260 nm.
Precipitate the collected material (~4 ml) with 2 ml of 7.5 M ammonium acetate and 16 ml ethanol. Resuspend the pellet in 120 p.l TE, wash the tube with 20 p,l TE and pool the 2 samples. This procedure provides haptenylated driver of the Human Fetal Brain cDNA library for use in normalization of the Human Fetal Brain cDNA
library.
Subtractive hybridization is carried out using the following procedure.
Denature a mixture of the following components at 80°C for 1 min: 1 ~g circular w ss cDNA library (see above), 0.5 pg of the oiigodA oligonucleotide S' (A)4° 3' (oligo dA), 3 pg of SP6 promoter-SaII sense oligonucleotide 5'GAA GGT ACG
CCT GCA GGT ACC GGT CCG GAA TTC CCG GGT CGA CCC ACG 3' (SEQ ID NO:1 ) (SP6-SaII), 0.25 M NaCI in 22 p.l of lx hybridization buffer [SO
mM HEPES (pH 7.5), 1 mM EDTA and 0.1 % SDS]. After denaturation, incubate the mixture at room temperature for 30 min.
For the COT=500, library denature 85 pg of the biotinylated RNA driver (see above) in 22 ~1 of 1X hybridization buffer at 90°C for 2 min, chill on ice for 1 min, and add 1 p.l of S M NaCI. Transfer the prehybridized circular ss DNA
to the biotinylated RNA driver and incubate at 42°C for 24 hr. For the COT=5 library, 10.5 p.g of RNA driver is hybridized for 2 hrs; for the COT=50 library, 41 ug of RNA driver is hybridized for 5 hrs; for the COT=0 library, no RNA
driver is added and the mixture is incubated for 24 hrs.
Following the incubation, transfer the mixture to a fresh tube, add 25 Eig of streptavidin and incubate at room temperature for 5 min. Extract the solution with an equal volume of PCIA (phenol/chloroform/isoamyl alcohol, 25:24: 1).
Back-extract the organic phase with 15 pl of TE containing 1 M NaCI and pool the aqueous extractions. Repeat the streptavidin binding and PCIA extraction «'O 99/15702 PCT/L1S98/19948 -:34-twice more. Precipitate the aqueous phase with () s M sodium acetate and ethanol.
Resuspend the pellet in 15 Irl '('F; and dialye against 'fE ( l Om.M:O.~rnM.) for 30 min. Transfer the DNA to a fresh tube and measure the volume. This resulting cDNA is a single-stranded normalized cDNA librarry.
Analysis of clones following subtraction is done in the following way.
When the circular ss cDNA that remains following subtraction is converted into ds cDNA using an oligodA-lVotl primer, dNTPs, a repair polymerase and is transformed into 1:;. coli cells, a large fraction of the transformants contain plasmids that do not contain inserts (fable 1 ).
Table I. Percent Recombinant cUNA ~;lones and Average ><nsert Size Following Total Human Fetal Brain cDNA Library Subtraction.
Human Fetal Brain % recombinants Average insert cDNA Libra 24 inde endent clones)size kb Cot 0 92 1.3 Cot=~5 79 1.2 Cot=50 6~ I .4 Cot=500 4 ~ 1.1 After analysis of the clones that do not contain inserts, it was determined that they were present in the original library at a frequency of less than 1 %, but were enriched following subtraction since they have no corresponding driver molecule to subtract there (Figure 2). 'I°wo approaches were developed to remove this form of background and are described in Examples 2 and 3.
Example 2. Refnooal of Background .from rr Nornralizerl cDNtl Library Using Selection mitl: a Target Sj~ecifc Biotinylated OligodA
2.~ Notl Probe As a result of the subtraction process described in Example l, there is a trend of increased background that depends directly on the COT of the subtraction step (Table 1 ). Since a total 9ibrary driver is use~3, clones that do not contain a V1'O 99/15702 PCT/L1S98/t994g _35_ :c>unterpart in the e.lriver will Ite. enricheGl ~dlttis wars ob=erved irt the process described it Example 1 (Figure 2:1. 'fo address this issue. two methods were developed and a third is described to virtually eliminate the background. In the first case, described in this example, sefectic~rt of recombinant clones using an oligodA-Notl biotinylated probe was used (Figure 3) as follows.
Following subtraction, repair and transformation, 45% of the clones derived from the CO'f=X00 protocol were recombinant ('table 1), however by using probe selection with a biotinylated oligodA-NotI primer (5'(A)r SGGG CGG
C'.CG C 3') (SEQ ID N0 2), the recombinant clones were selected away from the non-recombinants permitting construction of a normalized cDNA library with no significant change in average insert. size arid the virtual elimination of non-recombinant clones (Table 2).
Table Z. Percentage of Recombinant cDNA Clones and Average Insert Size Following Total Human Fetal Brain cDNA Library Subtraction and GENETRAPPERT~~ Selection with a Biotinylated OligodA-NotI Probe.
Human Fetal Brain /p recombinants Average insert eDNA Library (96 rode endent size kb clones Cot=500 ~ ~9 [ 1.2.5 [

More than 98% of the clones picked at random contain inserts that are on average as large as the non-normalized cl7NA library fiom which they were derived. In addition, PCR analysis of rar-t=: and abundant TGF-~i amplicons indicates that substantial norrrtalization has been accomplished (Figure G).
Note that although the TGF-(31 PCk. product is undetectable in the non-normalized and low COT libraries, it is detected in the higher Cot libraries.
2j The normalized circular ss cDNA from Example 1 was heated at 70°C
for 1 min and chilled on ice for 1 ntin. 200 ng of the biotinylated oligodA-Notl primer see above), was hybridized at :37"C: for 1 lu-. "hhe hybridization mixture was incubated with 80 p,g of streptavidin magnetic beads. The beads were marked three times with I 00 ltl 01 wash buffer ( 1 () tn~'~I T~ris~HC! [pt-I 7. S), 1 mM FDTA).

The beads were resuspended in 20 pl I X elution buffer I 0 mIVI glycine and the eluate was saved. The elution step was repeated with 1 J ul of 1 X elution buffer and the eluates were pooled. This protocol was repeated three times and the eluates.
S The captured single stranded cDNA was repaired as follows: Make a repair mix by combining 4 pl of l OX repair buffer [ 100 rrLTVI Tris-HCl (pH
8.8 at 25°C), 1 S mM MgCl2, 500 mM KCI, I% Triton X-100], 1 ~I 10 mM dNTP, I
p.l of repair enzyme Dynazyme (2 m/pl) (Thermus brockianr~s from Finnzymes) and 34 p.l of water. This mixture was mixed and stored on wet ice. A DNA primer mix was prepared by adding the following to a fresh microcentrifuge tube: 4 ~I
of 10X repair buffer, 35 pl of captured cDNA from the previous step and 1 p.l (~0 ng) of unbiotinylated oligo dA-NotI primer. The primer mix was centrifuged at room temperature for 2 sec at 14,000 x g and incubated at 95°C for 1 min. At the same time, the repair mix was incubated at 70°C. The DNA primer mix was transferred to the 70°C bath and incubated for 1 min. 40 pl of the prewarmed repair mixture was added to the tube containing the DNA primer mix. The contents were mixed by pipetting and then the mixture was incubated at 70°C for 15 min to allow primer extension (synthesis of double stranded cDNA). The tubes were removed from the water bath and centrifuged at room temperature for 2 s at 14,000 x g. The repaired DNA was precipitated by adding I p.l glycogen, 41 pl of 7.5 M ammonium acetate, and 320 ftl of -20°C ethanol to each tube. The tubes were vortexed and placed in ice for 10 min or at 4°C overnight.
The tubes were then centrifuged at 4°C for 30 min at 14,000 x g. The ethanol was carefully removed from the small pellet and layered with 100 pl of 70% ethanol (-20°C).
2~ The tubes were centrifuged at 4°C for 2 min at 14,000 x Q and all of the ethanol was removed and the pellets dried at room temperature for 10 min or until dry.
The pellets were dissolved in 10 p.I of TE buffer and store at 4°C. 2 p1 of aliquots ofthe repaired DNA was electroporated per 20 ul aliquots ofDHIOB ElectroMax Competent E. cull.

~\'O 99/I5702 PCT/CJS98/19945 -..i 7-E~-anrple 3. Removal oj- l3rrckl;;rourrrl _Ji-rrrn rr Nr~rrrrrrli~ecl cDNA I
ibrary>
Llcirz~~ UligorL9-Notl Rr fruir Synthesis with Nuclcotifle Analogues rvlrich Comer Nrrcleasce Resistance.
Using the approach in Example 2 to remove background, to construct a normalized cDNA library with greater than I x 10~ primary clones minimally requires three independent selections and I S electroporations (Table 3).
Table 3. Comparison of Various Methods to Remove Background.
Method Number of 'Cotal # of Electro ~oralionsclones recombinants Biotinylated probe1 S I .? X >95%

selection 3 selections ~

Nuclease resistant5 4.8 X 10~ >95%

repair selection To address this issue, an alternative approach was developed to reduce background in normalized Libraries. In this method, called nuclease resistant repair synthesis, the same probes described in example 2 is used, oligodA-A/otI, but in this case it is not biotinylated (Figure 5). However-, biotinylated probes as used in Example 2. may be used to include the additional selection step of Example 2.
~'~'hen compared to the selection method of Example 2, a library can be constructed that is four times as complex and requires one third the number of 2t7 electroporations (Table 3). In addition the library background is virtually eliminated acrd the insert size ofthe Library is unchanged (Table 4). Finally, when highly abundant genes were examined by colony hybridization, their abundance was decreased I S- to 18-fold (Table 5) and the abundance of rare genes was substantially increased (Figure fi).

W'O 99/15702 PC.'T/LJS98/19948 Tafile 4. Percent Recombinant cDNA C."loner and .W erage Insert Size Following Total Human Fet:rl Brain cI>NA I_,iUrarv Subtraction and -methylcytosine/Hlrrr( 'Treatment.
Human Fetal Brain % recombinants Average insert cDNA Libra (80 inde~aenderrt r_lones) size (kb) Cot==500 =~9 S 1 Table S. Normalized cDNA Library Analysis: Depletion of Abundant cDNAs Depends Directly Upon the Extent of Subtraction.
Gene (~ot=0 <'.ot=5 Cot=50 Cot=500 a-tubulin 0.78 ~0 0.62 ~o 0.24 % 0.043 EF-1 a O.~t'2, 0.28 ',r - 0.029 % 0.13 %

Colony hybridization using "P-labe~lerl vlit;onr.o:leot:ide probes directed to the a-tubulin and elongation factor 1 (EF'-1 a) Single-stranded normalized cDNA library l;enerated by subtraction (see Example 1) was repaired as follows: A repair mix was made by combining 3 p.l of 1 OX repair buffer [ 100 mM Tris-HCl (pH 8.8 at 25 °C), 15 mM MgCl2, 500 mM
KCI, 1% Triton X-100], I l~l 10 mM dNTI' (containing 10 mM 5 methyl dCTP), 1 p.l of repair enzyme Dynazyme (2 u/ul) (7herrnus broc~-ianus from F'innzymes) and 25 pl of water, mixing and storing on wet ice. A D\A primer mix for each reaction was made by adding the following to a fresh microcentrifuge tube: 11 pl autoclaved, distilled water, :3 pl of l OX repair buffer, 15 ~I of dialyzed DNA from the previous step, and I lO (50 ng) ofunbiotinylated oliQo A-NotL. The mixture was centrifuged at room temperature for 2 see at 14,000 x g. The DNA primer mix was incubated at 95°(, fcmr 1 min. At the same time, the repair mix was incubated at 70°C. The DhlA primer rnix w~.rs transferred to the 70°C bath and incubated for 1 min. 30 yl of the prewarmed repair mixture was added to the tube containing the primer mix. 'fhc contents were, mixed by pipetting and incubated at 70°C for 15 min to allow primer extension {synthesis of double stranded DNA).
The tubes were removed from tloe water bath ,zrfd centrifimed at morn temperature for 2 sec at 14,000 x a. The repaired DNA was przcipitated by adding 1 yl glycogen, 32 p.i of7.S M ammonium acetate, and 2S0 ul of-20°C ethanol to each tube- The tubes were vortexed and placed in ice for 10 min or at 4°C
overnight.
The tubes were then centrifuged at 4°C for 30 min at 14,000 x g. The ethanol was S carefully removed from the small pellet and layered with 100 ~.l of 70%
ethanol (-20°C). The tube was centrifuged at 4°C for 2 min at 14,000 x g. All of the ethanol was removed and the pellets at room temperature for I O min or until dry.
The pellets were dissolved in 10 p.l of TE buffer and store at 4°C. The repaired DNA was digested with 0.5 unit ofHhal in 20 pl of 1X buffer ( SmM Tris~HCI, pH 8.0; 1 mM MgCl2; 5 mM NaCI) at 37°C for 30 min. The DNA was ethanol precipitated and resuspend the dried pellet resuspended in 8 ~! of TE. 2 ftl aliquots of the repaired DNA was electroporated per 20 p.l aliquot of DH10B
ElectroMax competent E. coli.
Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

-3~7. I-SEQUENCE LI:.=,TINt:i <110> Life Technologies, Ir~c.
9800 Medical (.enter Driz~e Rockville, Maryland :~08's0-332,1 USA
<120> Normalized Nucleic Acid Libraries an<~ Met:hods of Production Thereof <:L30> 184-296 <_L40> 2, 304, 895 <:L41> 1998-09-24 <150> US 09/159,496 <151> 1998-09-23 <150> 60/059,817 <151> 1997-09-24 <160> 2 <170> PatentIn Ver. 2.0 <;?10> 1 <:?11> 48 <212> DNA
<:?13> Artificial sequence <''<?20>
<:?23> Description of artificial :sequence: synthetic oligonuc:leotide <400> 1 gaaggtacgc ctgcaggtac cggtccggaa ttcc:cgggtc gacccacg 48 <210> 2 <i?11> 25 <:? 12 > DNA
<:?13> Artificial sequence <:?20>
<:?23> Description of~ artif:icial sequence: ~y~nt~zetic oligonuc_Leotide <<~00> 2 aaaaaaaaaa aaaaagggc:g gccgc 25

Claims (44)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for normalization of a nucleic acid library comprising:

(a) synthesizing one or more nucleic acid molecules complementary to all or a portion of the nucleic acid molecules of said library, wherein said synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules;

(b) incubating said nucleic acid library with said haptenylated nucleic acid molecules under conditions favoring the hybridization of the more highly abundant molecules of said library with the haptenylated nucleic acid molecules; and (c) removing said hybridized molecules by hapten-ligand interactions, thereby producing a normalized library.

2. The method of claim 1, wherein said nucleic acid library is a cDNA library.

3. The method of claim 2, wherein the nucleic acid molecules of said cDNA
library are single-stranded.

4. The method of claim 2, wherein the nucleic acid molecules of the said cDNA
library are double-stranded.

5. The method of claim 2, wherein said cDNA library is produced by a method comprising incubating a population of mRNA molecules under conditions sufficient to produce a cDNA library from said population of mRNA molecules.

6. The method of claim 1, wherein said haptenylated nucleic acid molecules are RNA molecules.

7. The method of claim 1, further comprising reduction or removal of contaminating nucleic acid molecules from said library.

8. The method of claim 7, wherein said reduction or removal is performed before or after normalization of said library.

9. The method of claim 7, wherein the contaminating nucleic acid molecules are one or more vectors.

10. The method of claim 7, wherein said reduction or removal comprises incubating said library with at least one haptenylated probe.

11. The method of claim 10, wherein said probe hybridizes to nucleic acid molecules of said library.

12. The method of claim 11, wherein said probe is not capable of hybridizing to vector sequences of said library.

13. The method of claim 10, wherein the haptens of said haptenylated probe are used to isolate a normalized library having substantially reduced contaminating nucleic acid molecules, thereby producing a selected normalized library.

14. The method of claim 13, wherein said haptens are selected from the group of avidin, streptavidin, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, an enzyme-specific substrate, polymyxin B, endotoxin-neutralizing protein (ENP), Fe3+, a transferrin receptor, an insulin receptor, a cytokine receptor, CD4, spectrin, fodrin, ICAM-1, ICAM-2, C3bi, fibrinogen, Factor X, ankyrin, an integrin, vitronectin, fibronectin, collagen, laminin, glycophorin, Mac-1, LFA-1, .beta.-actin, gp 120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody, biotin and combinations thereof.

15. The method of claim 14, wherein said hapten is biotin.

16. The method of claim 13, wherein isolation comprises the use of a solid support comprising at least one ligand that binds said hapten.

17. The method of claim 13, wherein said selected normalized library is single-stranded.

18. The method of claim 17, further comprising incubating said single-stranded selected normalized library under conditions sufficient to render said molecules of said selected normalized library double-stranded.

19. The method of claim 18, wherein said conditions comprise incubating said single-stranded selected normalized library with one or more nucleotides, one or more polypeptides having polymerase activity and one or more primers.

20. The method of claim 19, wherein said one or more nucleotides are nucleotide analogues which confer nuclease resistance on said double-stranded molecules.

21. The method of claim 20, further comprising digesting a sample comprising said double-stranded molecules with a nuclease.

22. The method of claim 21, further comprising transforming one or more host cells with said double-stranded molecules.

23. The method of claim 18, further comprising transforming one or more host cells with said double-stranded molecules.

24. The method of claim 19, wherein said primers hybridize to molecules of the library but not to contaminating nucleic acid sequences.

25. The method of claim 24, further comprising transforming one or more host cells with said double-stranded molecules.

26. The method of claim 6, wherein said RNA molecules are produced by one or more RNA polymerases.

27. The method of claim 26, wherein said RNA polymerases are selected from the group of SP6, T7 and T3 RNA polymerases.

28. The method of claim 6, wherein said RNA molecules are produced with one or more promoters.

29. The method of claim 28, wherein said promoters are provided by one or more vectors or by one or more adapters.

30. The method of claim 29, wherein said promoters allow the synthesis of at least one RNA molecule from all or a portion of the nucleic acid molecules of said library.

31. The method of claim 1, wherein said removal comprises the use of a solid support comprising at least one ligand.

32. The method of claim 4, further comprising treating said double-stranded cDNA
library under conditions sufficient to render said molecules single-stranded.

33. The method of claim 32, wherein said conditions comprise degradation of one strand of said double-stranded molecules.

34. The method of claim 32, wherein said conditions comprise denaturing said double-stranded molecules.

35. The method of claim 33, wherein said degradation is accomplished with gene II
and Exonuclease III.

36. The method of claim 1, wherein said hybridisation conditions are selected from the group 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 from about 10 to 10,000;
(e) a COT from about 25 to 10,000;
(f) a COT from about 50 to 10,000;
(g) a COT from about 100 to 10,000; and (h) a COT of less than 10,000.

37. The method of claim 7, wherein said reduction or removal comprises incubating said library with at least one primer and at least one nucleotide which confers nuclease resistance under conditions sufficient to make double stranded nucleic acid molecules.

38. The method of claim 37, wherein said primer hybridizes to nucleic acid molecules of said library.

39.The method of claim 37, wherein said primer is not capable of hybridizing to vector sequences of said library.

40. The method of claim 37, wherein said nucleotide is a nucleotide analog.

41. The method of claim 40, wherein said nucleotide analog is a methylated nucleotide.

42. The method of claim 41, wherein said methylated nucleotide is 5-methyldeoxycytosine.

43. The method of claim 37, further comprising digesting said double-stranded nucleic acid molecules with one or more nucleases to produce digested molecules.

44. The method of claim 43, further comprising transforming one or more host cells with said digested molecules.