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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
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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.
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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);
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(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
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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
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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
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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
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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
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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.
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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.
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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
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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.
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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
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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
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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.
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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
CA 02406391 2002-11-07
w0 99/15702 PCTlUS98/19948
-16-
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
CA 02406391 2002-11-07
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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-
701
( 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
CA 02406391 2003-08-12
-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-
CA 02406391 2002-11-07
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_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
CA 02406391 2002-11-07
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PCT/US98/19948
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
CA 02406391 2002-11-07
WO 99/15702 PCT/US98I19948
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
CA 02406391 2003-08-12
-22-
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
CA 02406391 2002-11-07
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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'
CA 02406391 2002-11-07
WO 99/15702 PCT/L1898/19948
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.
CA 02406391 2002-11-07
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_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.
CA 02406391 2002-11-07
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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,
CA 02406391 2002-11-07
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
CA 02406391 2002-11-07
WO 99/15702 I'CT/US98/19948
-28-
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
CA 02406391 2003-08-12
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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
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-30-
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
CA 02406391 2002-11-07
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_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
CA 02406391 2002-11-07
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_ 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
1X
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
25
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
CA 02406391 2003-08-12
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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
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«'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
CA 02406391 2002-11-07
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).
CA 02406391 2003-08-12
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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.
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~\'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%
1 O6
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).
CA 02406391 2002-11-07
W'O 99/15702 PC.'T/LJS98/19948
-3 8-
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
CA 02406391 2003-08-12
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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.
CA 02406391 2002-11-07
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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
CA 02406391 2002-11-07
<:? 12 > DNA
<:?13> Artificial sequence
<:?20>
<:?23> Description of~ artif:icial sequence: ~y~nt~zetic oligonuc_Leotide
<<~00> 2
aaaaaaaaaa aaaaagggc:g gccgc 25
