Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF PRODUCING A SUBTRACTION LIBRARY
This application claims priority to United States Provisional Application
Serial
Number 60/041,688, filed March 24, 1997.
Field of the Invention
The present invention relates generally to the field of generation of cDNA
libraries,
and more specifically, to methods of generating subtraction libraries. ~ -
Background of the Invention
Methods have been described for obtaining information about gene expression
and
identity using so called "high density DNA arrays" or grids. See, e.g., M.
Chee et al.,
Science, 274:610-614 (1996) and other references cited therein. Such gridding
assays have
been employed to identify certain novel gene sequences, referred to as
Expressed Sequence
Tags (EST) (Adams et a., Science, 252:1651-1656 ( 1991 )). A variety of
techniques have
also been described for identifying particular gene sequences on the basis of
their gene
products. For example, see International Patent Application No. W091/07087,
published
May 30, 1991. In addition, methods have been described for the amplification
of desired
sequences. For example, see International Patent Application No. W091/17271,
published
2 0 November 14, 1991.
Currently available subtraction techniques remove unwanted sequences from a
given library. In one approach, a large number of unknown genes are used to
drive the
subtraction library to remove the unknown genes from a library of interest.
See, e.g., J.
Love and P. Deininger, BioTechniques, 11 ( 1 ):88-92 ( 1991 ). However, while
this technique
is useful in removing a large number of genes from the library, little is
known about the
genes iii the resulting subtraction library, other than their source. In
another common
approach to subtraction, a small number of known genes {e.g., less than 100)
are used to
drive the subtraction to remove these sequences from the library of interest.
This approach
allows one to control which genes are removed, but current methods only permit
this
3 0 approach to be used with a small number of genes and the resulting
subtraction library
contains a significant amount of genes which are not desired.
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Accordingly, there exists a need for more efficient methods for producing
subtraction libraries. Also needed are more efficient methods for screening
for novel
pharmaceutical reagents.
Summary of the Invention
In one aspect, the present invention provides a method of producing a
subtraction
library utilizing a collection containing known or defined sequences. The
method involves
the steps of providing a surface having a collection of defined nucleic acid
sequences at~d
allowing this surface to come into association with a library containing
undefined nucleic
acid sequences under conditions which permit hybridization. The non-hybridized
nucleic
acid sequences are recovered, isolated, and form the subtraction library. The
subtraction
library is characterized by containing the undefined sequences from the second
library
which are not present in the first library of defined sequences.
In another aspect, the present invention provides a subtraction library
produced
according to the method of the invention.
In yet another aspect, the present invention provides a method of rapidly
screening
a library containing undefined sequences for the presence of known or defined
sequences
using the method steps described herein.
In still another aspect, the present invention provides a method of rapidly
screening
2 0 a library containing undefined sequences using a polynucleotide probe from
a known or
defined sequences using the method steps described herein.
Other aspects and advantages of the present invention are described further in
the
following detailed description of the preferred embodiments thereof.
2 5 Detailed Description of the Invention
The present invention provides a method of producing a subtraction iibrary
utilizing a collection of known or defined sequences. The method involves
providing a
surface having immobilized thereon a collection consisting of defined or known
nucleic
acid sequences, which is subsequently contacted with a library containing
unknown or
3 0 undefined sequences under appropriate hybridization conditions. Any non-
hybridized
polynucleotide, preferably DNA, is recovered as the subtraction library which
contains
sequences which were present in the library, and which differ from the
sequences of the
collection. Advantageously, the method of the invention permits a large number
of defined
2
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sequences to be used to drive a subtraction. Thus, this method permits the
efficient
production of a subtraction library whose content can be readily controlled.
Also described is a subtraction library prepared according to the method of
the
mvent~on.
I. Definitions
Several words and phrases used throughout this specification are defined as
follows: ' .:
As used herein, the term "gene" refers to the genomic nucleotide sequence from
which a cDNA sequence is derived. The term gene classically refers to the
genomic
sequence, which upon processing, can produce different RNAs.
By "gene product," it is meant any polypeptide sequence, peptide or protein,
encoded by a gene.
As used herein, "collection comprising known sequences," refers to any ordered
set
of nucleotide sequences, including RNA and DNA sequences, which may be in the
form of
plasmids, cDNA, PCR products, genes, gene fragments, DNA fragments,
oligonucleotides
and the like. Preferably, such known sequences within the collection have been
previously sequenced and/or are of known origin. Desirably, this collection
contains a
large number of sequences, e.g., as many as 100,000 - 200,000 members where
the
sequences are genes, or as many as 1,000,000 if the members of the collection
include
oligonucleotides. However, the number of sequences in the invention may be
varied as
desired and are not a limitation on the present invention. This collection may
contain
sequences drawn from a number of different sources, including a variety of
libraries. For
example, the collection may be drawn from one or more tissue source libraries
of a member
2 5 of the mammalian species, e.g., a human. Desirably, the human is healthy;
however,
libraries derived from a diseased or impaired individual may also be utilized.
Further, other
mammals of interest include, without limitation, a non-human primate, a
rodent, and a
canine. In a particularly prefened collection, defined sequences are present
in a single
copy.
3 0 When utilizing gene sequences in the methods of the invention, it is not
necessary
that the full-length sequence of the gene be known. Rather, all that the
methods of the
. invention require is that the portion of the gene sequence that renders it
unique is known,
which is approximately 17 base pairs. By "known nucleic acid sequence," it is
meant that
the sequence is reasonably unique and contains no redundancies or repeats.
3
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The term "library" includes, but is not limited to, plasmid libraries, RNA
libraries,
DNA libraries such as those containing PCR products from genomic libraries,
cDNA
libraries, oligonucleotide libraries and known sequences. Methods for the
construction of
such libraries are well known by those skilled in the art. A library may be
adjusted to
minimize the number of complete genes present in a single insert to
approximately one
gene. Techniques for this adjustment are well known to the skilled artisan.
"Isolated" means altered "by the hand of man" from its natural state; i.e.,
that, if it
occurs in nature, it has been changed or removed from its original
environment, or both.
For example, a polynucleotide or a polypeptide naturally present in a living
animal in its
natural state is not "isolated," but the same polynucleotide or polypeptide
separated from
the coexisting materials of its natural state is "isolated," as the term is
employed herein.
For example, with respect to polynucleotides, the term isolated means that it
is separated
from the chromosome and cell in which it naturally occurs.
As used herein, the term "solid support" refers to any substrate which is
useful for
the immobilization of a plurality of defined materials (i.e., sequences)
derived from a
library by any available method to enable detectable hybridization of the
immobilized
polynucleotide sequences with other polynucleotides in the sample. Among a
number of
available solid supports, one desirable example is the support described in
International
Patent Application No. W091/07087, published May 30, 1991. Examples of other
useful
2 0 supports include, but are not limited to, nitrocellulose, nylon, glass,
silica and Pall
BIODYNE C membrane. It is also anticipated that improvements yet to b;. made
to
conventional solid supports may also be employed in this invention.
The term "grid" means any generally two-dimensional structure on a solid
support
to which the defined materials of a library are attached or immobilized.
2 5 As used herein, the term "predefined region" refers to a localized area on
a surface
of a solid support on which is immobilized one or multiple copies of a
particular amplified
gene region or sequence and which enables hybridization of that clone at the
position, if
hybridization of that clone to a sample polynucleotide occurs.
By "immobilized," it is meant to refer to the attachment of the genes or other
3 0 nucleic acids to the solid support. Means of immobilization are known and
conventional to
those of skill in the art, and may depend on the type of support being used.
By "label" as used herein is meant any conventional molecule which can be
readily
attached to or incorporated onto RNA or DNA and which can produce a detectable
signal,
the intensity of which indicates the relative amount of hybridization of the
RNA to the
4
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DNA fragment or oligonucleotide on the grid. Preferred labels are fluorescent
molecules or
radioactive molecules. A variety of well-known labels can be used.
The term, "subtraction library," as used herein refers to a library that is
highly
enriched for novel nucleotide sequences, including novel genes and gene
fragments, among
other sequences.
II. The Collection of Known Seauences
In the practice of this method, one or more grids is prepared, so that each
grid
carries on its solid surface nucleotide sequences from a collection of defined
sequences
immobilized on the surface. Desirably, this collection is as defined above and
these
nucleotide sequences are, e.g., genes, gene fragments, other DNA fragments, or
oligonucleotide sequences.
Nucleotide sequences from the selected collection are gridded onto a surface
of a
solid support. Desirably, the nucleotides are in the form of, but are not
limited to, DNA,
which are put down on the surface in an amount between about 100 pg to about
1000 ng per
spot depending on substrate. In a further preferred embodiment the amount of
polynucieotide used is between about 10 ng to about 100 ng per spot. However,
RNA may
be utilized in similar amounts. Although not required, it may be desirable to
provide
duplicate or multiple coverage of the genes or other nucleotide sequences on
the surface.
The nucleotide sequences include, but are not limited to, individual clones
spotted onto and
2 0 grown on a surface of the solid support; or plasmid clones isolated from
said library, PCR
products derived from the plasmid clones, or oligonucleotides derived from
sequencing of
the plasmid clones, which are immobilized to the surface of the solid support.
Numerous conventional methods are employed for immobilizing these nucleotide
sequences to surfaces of a variety of solid supports. See, e.g., Affinity
Techniques, Enzyme
2 5 Purification: Part P, Methods in Enzymology, Vol. 34, ed. W.B. Jakoby, M.
Wilcheck,
Acad. Press, NY (1971); Immobilized Biochemicals and Affinity Chromatography,
Advances in Experimental Medicine and Biology, Vol. 42, ed. R. Dunlap, Plenum
Press,
NY (1974); U.S. Patent 4,762,881; U.S. Patent No. 4,542,102; European Patent
Publication
No. 391,608 (October 10, 1990); or U.S. Patent No. 4,992,127 (November 21,
1989).
3 0 Although not required, it may be desirable to immobilize the gene or other
nucleic
acid sequences in an array, such that the sequences are placed at predefined
locations or
regions on the surface. Knowing how the sequences are arrayed gives the
methods of the
invention a level of efficiency beyond that which is capable using the prior
art methods.
This is an important feature of the invention and it allows ready access to
the desired
5
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sequence, and preferably its related clone and associated data, following
screening. One
desirable method for attaching these materials to a solid support is described
in
International Application No. PCT/US90/06607 (published May 30, 1991 ).
Briefly, this
method involves forming predefined regions on a surface of a solid support,
where the
predefined regions are capable of immobilizing the materials. The method makes
use of
binding substrates attached to the surface which enable selective activation
of the
predefined regions. Upon activation, these binding substances become capable
of binding
and immobilizing the materials derived from the collection of defined
sequences. .:
Any solid substrates suitable for binding nucleotide sequences on the surface
thereof for hybridization and methods for attaching nucleotide sequences
thereto may be
employed by one of skill in the art according to the invention. Currently,
however, the
preferred surface is glass. As with other solid substrates, methods for
depositing and
binding nucleotide sequences to a glass surface are well known to those of
skill in the art.
See, e.g., L. A. Chrisey, et al., "Covalent Attachment of Synthetic DNA to
Self-Assembled
Monolayer Films", Nucleic Acids Res., 24:3031-3039 (1996); Silicon Compounds:
Register and Review (United Chemical Technologies, Inc., Bristol,
Pennsylvania, 1993). In
an alternative embodiment of the invention, the surface may be beads. Because
it is not
necessary that the surface be flat, a vertical surface is contemplated to be
within the scope
of the invention. Such a surface may have steps, ridges, kinks, terraces, and
the like.
III. The Library Comprising Undei~ined Seaaences
Once the grid surface containing the immobilized sequences from the collection
is
prepared, it is allowed to associate with sequences derived from a library
containing
undefined sequences under suitable hybridization conditions. Such a library
may include
2 5 sequences of unknown origin and/or unknown nucleotide sequences.
The library which provides the source of the unknown or undefined genes, gene
fragments, or other nucleic acid sequences may be a random cDNA library
obtained using
known techniques. Alternatively, a library of genes from a selected organ or
tissue, or a
mixed set of RNAs, may be the source of the sequences. Suitably, for use in
the method of
3 0 the invention, RNA is isolated and reverse transcribed to cDNA using
standard procedures
for molecular biology such as those disclosed by Sambrook et al., MOLECULAR
CLONING, A LABORATORY MANUAL, 2nd Ed; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor Lab Press, Cold Spring Harbor, NY 1989. The cDNA library is
then
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constructed in accordance with procedures described by Fleischmann et al.,
Science,
269:496-512 (1995). For the purposes of the present invention, the library
comprising the
undefined sequences may be a library, as is defined above. Most desirably,
however, the
. library is a plasmid library.
In a preferred embodiment, the source of the unknown or undefined sequences is
a
phagemid library composed of human DNA, and preferably, human cDNA. This
embodiment is particularly advantageous for isolation of the non-hybridizing
sequences
which compose the subtraction library produced by the method of the invention.
'Such a
phagemid library can be prepared using conventional techniques. See, e.g.,
Sambrook et
al., supra. Suitable vectors for use in this system include pBluescript
[Stratagene, La Jolla,
CA] and pUC118 [J. Vieira and J. Messing, Methods in Enzymolo~y, 153:3
(1987)]. Other
suitable vectors are well known and may be readily selected by one of skill in
the art.
As discussed above, however, other suitable techniques and vectors may be
utilized
to prepare the undefined library. For example, a phage library may be produced
using a
vector such as M 13; however, other suitable vectors are known in the art.
Optionally, these undefined sequences may be labeled to permit detection of
DNA
which hybridizes to the immobilized sequences. Known conventional methods for
labeling
the sequences may be used. For example, fluorescence, radioactivity,
photoactivation,
biotinylation, energy transfer, solid state circuitry, and the like may be
used in this
2 0 invention.
Desirably, the single stranded DNA is isolated from the library containing the
undefined sequences using conventional techniques. For example, where the
library is a
phagemid library, single stranded copies of the library are packaged into
particles using a
suitable helper phage. The resulting phage particles are then isolated by
convention
2 5 techniques, which typically involve centrifugation and precipitation. Such
techniques for
isolation of single stranded DNA are well known to those of skill in the art
and are not a
limitation on the present invention. Although less desirable, double stranded
DNA may be
utilized in the method of the invention.
3 0 IV. Hybridization to the grids
The isolated polynucleotide, preferably DNA (e.g., cDNA), obtained from the
undefined library is permitted to come into association with the immobilized
sequences
derived from the defined driver library under conditions which permit
hybridization.
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Preferably, hybridization takes place under stringent conditions, e.g.,
conditions
such that only sequences which are more than about 90% identical will remain
hybridized
throughout the procedures. However, if desired, other less stringent
conditions may be
selected. For example, less stringent conditions may be desired in order to
increase
hybridization, thereby decreasing the size of the subtraction library. Thus, a
first
hybridization may be performed at very low stringency, permitting a
significant amount of
hybridization and producing a very small subtraction library. Subsequent
washes increased
or increasing stringencies may then be used to control the size and content of
the
subtraction library.
Techniques and conditions for hybridization at selected stringencies, such as
those
described herein, are well known in the art. See, e.g., Sambrook et al.,
Molecular Cloning.
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (
1989).
It is preferred that multiple rounds of hybridization are carried out,
preferably using
the conditions set forth elsewhere herein. It most preferred that the number
of rounds of
hybridization be between about 3 and 6.
V. Isolation of Non-Hybridized DNA
Following completion of hybridization, the "undefined" sequences which did not
hybridize to the immobilized sequences on the grid surface are recovered and
purified away
2 0 from the hybridization solution using standard techniques. See, e.g.,
Sambrook et al.,supra.
Iu a preferred embodiment, wherein the DNA is produced using the phagemid
system, the isolated, non-hybridized DNA is converted to double-stranded
plasmid by
synthesizing the second strand of the non-hybridized DNA. The resulting
plasmid is
propagated under suitable conditions in an appropriate cell. Suitable host
cells may be
2 5 readily determined by one of skill in the art, taking into consideration
the type of plasmid
utilized. Desirably, however, the host cells are selected from among bacterial
cells.
Currently, the preferred bacterial host is an E. coli strain. Following cell
culture, the DNA
from any resulting colonies is isolated using conventional techniques.
In another embodiment, by utilizing labelled sequences, the method of the
3 0 invention also permits rapid identification of sequences which the library
has in common
with the immobilized sequences from the collection. Particularly, according to
the
invention, upon hybridization one can readily detect labelled sequences, and
thereby
identify those sequences which are being removed from the library and will not
be
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contained in the subtraction library. The detected sequences are those which
are common
to the undefined library and the collection of defined sequences.
The collection of DNA isolated from the colonies forms the subtraction library
of
the invention. This subtraction library is characterized by containing DNA
sequences
present in the library containing undefined sequences, but excludes those
sequences which
were present in the collection of defined sequences which was immobilized on
the grid
surface. Techniques for maintaining these subtraction libraries are well known
to those of
skill in the art.
The DNA in the resulting subtraction library may be sequenced using standard
protocols [Sambrook et al., supra or ABI Prizm sequencing kit, Foster City,
CA] or utilized
for a variety of other purposes.
VI. Subtraction library
Thus, the present invention provides a subtraction library produced according
to the
method of the invention. This subtraction library provides a source of novel
nucleotide
sequences, including novel genes and gene fragments, among other sequences.
These
sequences and, particularly the genes and gene fragments, may be useful in
screening drug
candidates. The information generated thereby can be used in the
pharmaceutical industry
to identify new drugs.
2 0 Further, these sequences may be employed in conventional methods to
produce
isolated proteins or peptid.,s encoded thereby. To produce a protein or
peptide of this
invention, the polynucleotide sequences, preferably DNA, of a desired gene of
the
invention or portions thereof identified by use of the methods of this
invention are inserted
into a suitable expression system. In a preferred embodiment, a recombinant
molecule or
2 5 vector is constructed in which the polynucleotide sequence encoding the
protein or peptide
is operably linked to a heterologous expression control sequence permitting
expression of
the human protein. Numerous types of appropriate expression vectors and host
cell systems
are known in the art for mammalian (including human), insect, yeast, fungal
and bacterial
expression.
3 0 The transfection of these vectors into appropriate host cells, whether
mammalian,
bacterial, fungal or insect, or into appropriate viruses, results in
expression of the selected
proteins. Suitable host cells, cell lines for transfection and viruses, as
well as methods for
construction and transfection of such host cells and viruses are well-known.
Suitable
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methods for transfection, culture, amplification, screening and product
production and
purification are also known in the art.
In one embodiment, the nucleotides and proteins or peptides encoded thereby
which have been identified by this invention can be employed as diagnostic
compositions
useful in the diagnosis of a disease or infection by conventional diagnostic
assays. For
example, a diagnostic reagent can be developed which detectably targets a
nucleotide
sequence or protein of this invention in a biological sample of an animal.
Such a reagent
rnay be a complementary nucleotide sequence, an antibody (monoclonal,
recomb'inant.or
polyclonal), or a chemically derived agonist or antagonist. Alternatively, the
nucleotides of
this invention and proteins or peptides encoded thereby, fragments of the
same, or
complementary sequences thereto, may themselves be used as diagnostic
reagents. These
reagents may optionally be detectably labeled, for example, with a
radioisotope or
colorimetric enzyme. Selection of an appropriate diagnostic assay format and
detection
system is within the skill of the art and may readily be chosen without
requiring additional
explanation by resort to the wealth of art in the diagnostic area.
Additionally, genes and proteins or other sequences identified according to
this
invention may be used therapeutically. For example, nucleotides or proteins
or, peptides
identified using the subtraction library of the invention may serve as targets
for the
screening and development of natural or synthetic chemical compounds which
have utility
2 0 as therapeutic drugs. Alternatively, compounds which inhibit expression of
a gene or
protein are also believed to be useful .herapeutically. In addition, compounds
which
enhance the expression of genes essential to an organism may also be used.
Conventional assays and techniques may be used for screening and development
of
such drugs. For example, a method for identifying compounds which specifically
bind to
2 5 or inhibit proteins encoded by these nucleotide sequences can include
simply the steps of
contacting a selected protein or gene product with a test compound to permit
binding of the
test compound to the protein; and determining the amount of test compound, if
any, which
is bound to the protein. Such a method may involve the incubation of the test
compound
and the protein immobilized on a solid support. Still other conventional
methods of drug
3 0 screening can involve employing a suitable computer program to determine
compounds
having similar or complementary structure to that of the gene product or
portions thereof
and screening those compounds for competitive binding to the protein.
Identical
compounds may be incorporated into an appropriate therapeutic formulation,
alone or in
combination with other active ingredients. Methods of formulating therapeutic
CA 02284481 1999-09-21
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compositions, as well as suitable pharmaceutical carriers, and the like are
well known to
those of skill in the art.
Accordingly, through use of such methods, the present invention is believed to
provide compounds capable of interacting with these genes (or other nucleotide
sequences),
or encoded proteins or fragments thereof, and either enhancing or decreasing
the biological
. activity, as desired. Thus, these compounds are also encompassed by this
invention. ,
VII. Subtracted Probes
Further, the present invention provides probes produced by subtraction
according to
the method of the invention. These probes may be obtained, for example, using
the
following method, as well as using methods described elsewhere herein.
Subtracted probes
may be made by subtracting known genes from a mixture of unknown
polynucleotides,
preferably DNA. Following rounds of subtraction as set forth in the invention,
the
remaining polynucleotides are the probes for probing unknown polynucleotides,
such as by
the hybridization methods of the invention. These probes may be labeled using
well known
methods, such as colorimetric labeling or radiolabeling. These subtraction
probes provide a
source of novel nucleotide sequences, including novel genes and gene
fragments, among
other sequences, that are particularly useful to detect unknown polynucleotide
sequences,
especially in mixtures and libraries, gridded or in solution. These probe
sequences and,
2 0 particularly the genes and gene fragments, may be useful in screening
target candidates for
drug screening. The information generated thereby can be used in the
pharmaceutical
industry to identify new drugs.
Subtracted probes, may also be used to prime polynucleotide amplification
reactions, such as PCR. To achieve this, the subtracted probes could be made
using
2 5 degenerate polynucleotides sequences comprising a priming site on at least
one end of the
molecule. Two priming sites may also be added to the molecule, one at each
end; and these
may be the same or different sequences. The priming site, for example a known
sequence
between about 5 and 40 nucleotides in length, may be added to the subtracted
probes using
ligase. These subtracted probes may be used as primers for amplification in
solution or on
3 0 a solid support. In a preferred method, these primers are hybridized to a
collection of
polynucleotides on a solid support. Following hybridization, double stranded
- polynucleotide, such as double stranded DNA, could be produced by
amplification,
preferably by PCR. The polynucleotide primers useful for amplification
comprise
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sequences that is complementary to the priming site, or sites, where there is
more than one
and they are different. The amplified fragments may be cloned, for example,
using any
known method or as described herein, such as using the zero blunt end kit from
InVitrogen
(Carlsbad, CA).
Further, these probe sequences may be employed in conventional methods to
obtain
genes by hybridization which genes may be used produce isolated proteins or
peptides
encoded thereby. Methods described elsewhere herein may be used to produce a
protein or
peptide of this invention. ' .:
In one embodiment, the probe nucleotides identified by this invention can be
employed as diagnostic compositions useful in the diagnosis of a disease or
infection by
conventional diagnostic assays as described elsewhere herein or known in the
art.
These examples illustrate the preferred methods of the invention. These
examples
are illustrative only and do not limit the scope of the invention.
Example 1 - Preparation of a Subtraction Library
A. Immobilization of Collection of Known cDNA Seguences
The driver collection, which contains defined cDNAs, is gridded onto a
solid glass surface as follows. The collection is engineered into the
pBluescript Vector
2 0 (Stratagene, La Jolla, CA) and inserts are recovered via PCR using vector-
specific primers.
The i~~serts containing the cDNA are deposited on the glass surface via
microcapillaries
and attached using standard techniques [Silicon Compounds: Register and Review
(United
Chemical Technologies, Inc., Bristol, Pennsylvania, ( 1993}].
B. Generation of Undefined cDNA Library
2 5 The undefined cDNA library from the desired source is constructed using
the Superscript Plasmid system (Life Technologies, Gaithersburg, MD) according
to
manufacturer's protocol with the exception that a modified vector is
substituted. Briefly,
pUCI 18 [J. Vieira and J. Messing, Methods in Enzymology, 153:3 (1987)] is
modified to
contain the desired cloning sites, and to remove sequences present in the
pBluescript
3 0 multiple cloning site to avoid spurious hybridization. Deletion of the
undesired sequences
may be performed using Quick Change Mutagenesis kit (Stratagene, La Jolla,
CA),
according to manufacturer's protocol or another conventional method. The
vector has
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previously been engineered to contain sequences which permit isolation of
single stranded
DNA packaged as a bacteriophage, i.e., a phagemid vector.
C. Subtraction
Single stranded DNA is isolated from the library to be subtracted (i.e., the
cDNA library of B) using a helper phage according to methods described in
Sambrook et
al.> cited above. The single stranded library is hybridized to the gridded
library of Part A.
using 4x SSC, 42°C, 16-48 hours, at 20 pls. After the appropriate
hybridization period, the
hybridization solution is recovered, and precipitated using standard
techniques {Sambrook
et al.,supra). In a preferred embodiment, multiple rounds of hybridization are
carried out,
preferably using the conditions of this Example 1C. It is most preferred that
the number of
rounds of hybridization be about 4.
Alternatively, hybridization may be performed at a lower temperature, e.g.,
37°C. After hybridization, the hybridization solution is first
collected and then treated as
below. The grid is then washed with 2X SSC at 37°C, the wash collected,
and then
precipitated. The DNA is recovered as described below. These steps may then be
repeated
at a higher temperature, e.g., 65°C, and then with 0.2 X SSC at both
temperatures.
Following hybridization, the precipitated DNA is converted to double
stranded DNA using standard procedures {Sambrook et al., cited above.).
Briefly, a vector-
specific oligonucleotide is hybridized to the library, followed by synthesis
of the second
2 0 strand by E. coli DNA polymerase in the presence of T4 DNA ligase to
complete the
reaction.
The resulting double stranded DNA is electroporated into an appropriate E.
coli host strain (e.g., DHSalpha, Life Sciences Technology, Gaithersburg, MD)
and the
resulting colonies are harvested, forming the subtraction library.
All publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their entirety as
if each individual publication or reference were specifically and individually
indicated to be
incorporated by reference herein as being fully set forth. Any patent
application to which this
3 0 application claims priority is also incorporated by reference herein in
its entirety in the
manner described above for publications and references.
13
_. ___~ ._. . _ __ __ _._.____ _T _.
CA 02284481 1999-09-21
WO 98/43088 PCT/US98/05644
The above description fully discloses the invention, including preferred
embodiments thereof. Modifications and improvements of the embodiments
specifically
disclosed herein are within the scope of the following claims.- Without
further elaboration,
it is believed that one skilled in the art can, using the preceding
description, utilize the
present invention to its fullest extent. Therefore, the examples provided
herein are to be
construed as merely illustrative and are not a limitation of the scope of the
present
invention in any way. The embodiments of the invention in which an exclusive
property or
privilege is claimed are defined as follows. ~ ._
14
