JP2002527118A - Method for manipulating a complex nucleic acid population using peptide-labeled oligonucleotides - Google Patents

Abstract

(57) SUMMARY The present invention relates generally to methods for labeling, selecting, and screening a population of nucleic acids. In particular, the present invention relates to methods for selecting and comparing complex nucleic acid populations, such as cDNA libraries. These nucleic acid complex populations can be derived from cell or tissue types with various phenotypes of potential clinical interest. The method generally, ValiGene ^SM peptide tag known as oligonucleotides (P eptide- L abeled O ligonucleotide) method or VG-PLO ^SM, identifiable and identifiable peptide tag linked to the same oligonucleotide primers Including the use of

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] 1, TECHNICAL FIELD THE INVENTION The present invention relates generally to a population of nucleic acid, labeling, sorting, comparing, and a method of isolating. In particular, the present invention relates to a method for selecting and comparing a nucleic acid complex population such as a cDNA library. These nucleic acid complex populations can be derived from cells, cell types or tissue types with phenotypic variations that can be of clinical interest. The method generally comprises:
ValiGene ^SM peptide-labeled oligonucleotide (P eptide- L abeled O ligonucleotide
) Or manipulating the nucleic acid using an identifiable and identifiable peptide tag linked to an oligonucleotide primer, called VG-PLO ^SM .

[0002] 2. BACKGROUND OF THE INVENTION Labeled oligonucleotides have been used to detect specific sequences. For example, Bu
rdick and Oakes (Nucleic acid detection diagnostic kit and diagnostic method using solid-phase capture means,
European Patent Publication No. EP 0370 694 A2, published on May 30, 1990),
Disclosed is the use of oligonucleotide primers having a specific nucleic acid sequence that is complementary to a predetermined sequence of interest. The method is limited to the identification of a known sequence in a given sample, and each primer pair must correspond to a single, given PCR product.

[0003] Currently, several gene expression assays are becoming feasible to quantify the effect of drugs on the expression of most genes and proteins in cell culture.
(E.g., Schena et al., 1995, Quantitative monitoring of gene expression patterns using complementary DNA microarrays, Science 270: 467-470; Lochort et al., 1996, Monitoring expression by hybridization to high-density oligonucleotide arrays,
Nature Biotechnology 14: 1675-1680; Blanchard et al., 1996, Sequencing on Arrays: Exploring the Mystery of the Genome, Nature Biotechnology 14,1649; 1996, October 1996
See US Patent No. 5,569,588, issued to Ashby et al. On 29th, entitled "Methods for Drug Screening"). Raw data obtained from these gene expression assays is often difficult to interpret logically. Such measurement techniques typically detect many genes whose expression changes in response to the drug. Usually 50 to 100, but can be as high as 1,000 or as few as 10.

In some, but not all, of the phenotypes of interest,
Or, there are few methods for rapidly comparing multiple mixtures of nucleic acids to select for commonly expressed genes. Thus, there is a need in the art for a method that allows for a quick and effective comparison between nucleic acid populations.

[0005] 3. SUMMARY OF THE INVENTION The present invention provides methods for labeling, selecting, comparing, and screening multiple nucleic acid complex populations. These populations may be cDNA libraries constructed from phenotypically distinguishable cell or tissue types of interest. The method is extremely flexible and can be adapted to perform many complex sorting and comparison tasks.

[0006] The methods of the present invention may also be used to increase and supplement the analytical capabilities of other techniques for manipulating complex cDNA populations. A major advantage of the method of the present invention is that multiple cDNA
The ability to screen populations (eg, from different tissues belonging to the same individual, or from different phenotypic cell types that are simultaneously present in a given tissue sample).

[0007] The present invention has the advantage that multiple nucleic acid populations can be handled by various selection and molecular comparison methods. The present invention uses oligonucleotide primers having a peptide tag that can be identified and identified. Using the primers, a PCR reaction can be initiated from the sequence of the vector. Using such oligonucleotide primers, inserts from any given cDNA library can be labeled with a library-specific peptide tag. An identifiable tag serves to identify the library from which any given insert is derived. In addition, the identifiable tags allow the inserts to be selectively sorted and isolated based on the library from which they are derived, regardless of the complexity of the mixture of products. For example, a chromatography matrix having an antibody specific for one of the distinguishable peptide tags can be used. Such a matrix is capable of capturing or retaining both single-stranded and double-stranded fragments having a specific peptide tag. The fragment without this tag remains free in the effluent.

The method of the present invention uses a polymerase chain reaction (PCR) to first
Linearize all inserts in the library and add an identifiable and identifiable peptide label to all inserts from a particular cDNA library to indicate the library from which it was derived Can be. PCR can be used at various stages to amplify a complex mixture of products.

[0009] Nucleic acid sample populations can be from many different sources. Such sources include different phenotypes that are simultaneously present in a given tissue sample, or different tissues belonging to the same individual. One phenotype may be a "healthy" condition and the other phenotype may be a typical pathological condition, but this is not required. By the method of the present invention, genes that are specifically expressed in relation to each phenotype are identified, and genes that are expressed independently of the phenotype are compared, or several, but not all, of the phenotypes It is possible to compare the expressed genes.

In a first embodiment, the invention comprises the following steps: (a) using DNA from each of a plurality of cDNA libraries by PCR using oligonucleotide primers having a label specific to each library. (B) first in step (a)
The DNA labeled with the above-mentioned label is different from the DN labeled with the above-mentioned label in step (a).
A); and (c) selecting the DNA contacted in step (b) using one or more molecules each capable of binding to a label specific to each library. provide.

[0011] The present invention further provides, in a first embodiment, a further method wherein the label specific to each library is a 5 'peptide label.

[0012] The invention further provides, in a first embodiment, a further method wherein the label specific to each library is biotin.

[0013] The invention further provides, in a first embodiment, a further method, wherein said one or more molecules is an antibody.

The invention further provides, in a first embodiment, that the PCR is initiated from a vector sequence common to the nucleic acids in a particular library by oligonucleotide primers (
Therefore, for each library, use different primers), or oligonucleotide primers initiate PCR from sequences of vectors common to multiple cDNA libraries (therefore, such multiple cDNAs). PCR is performed using the same oligonucleotide primers for all libraries).

The present invention further provides, in a first embodiment, the following steps: (d) step
denaturing the resulting hybrid DNA strand of (b); (e) contacting the denatured single strand with the single-stranded binding protein to avoid reannealing the strand; and (f) contacting the single-strands coated with the single-strand binding proteins formed in (e) with one or more molecules each capable of binding to one of the labels specific to each library. Provide a sorting method.

[0016] The invention further provides, in a first embodiment, a method wherein at least one of the one or more molecules in (e) is an antibody.

The present invention provides in a second embodiment the following steps: (a) labeling DNA from the first cDNA population by PCR using an oligonucleotide primer having a first 5 ′ peptide label (B) labeling DNA from the second cDNA population by PCR using oligonucleotide primers having a second 5 'peptide label; (c) under conditions in which hybridization can occur, step (a) )) Contacting the DNA labeled in step (b) with the DNA labeled in step (b); and (d) converting the DNA having only the first or second 5 ′ peptide label from the first and second 5 ′ peptide DNA with label
And a method for comparing cDNA libraries.

The invention further provides, in a second embodiment, the first population of cDNAs is derived from one or more cells or organisms that have been placed in the first state, and the second population of cDNAs is the first population of cDNAs. Further methods are provided, wherein the method is derived from one or more cells or organisms of the same type which have not been placed in a condition.

The present invention further provides, in a second embodiment, the first population of cDNAs is derived from one or more cells or organisms placed in the first state, and the second population of cDNAs is in the second state. A further method is derived from one or more cells or organisms of the same type as described above, which are located at

The present invention further provides, in a second embodiment, the first and second cDNA populations:
Further methods are provided wherein the phenotype is from a different cell or organism.

The present invention further provides, in a second embodiment, a nucleotide sequence of an oligonucleotide primer pair having a first 5 ′ peptide label and an oligonucleotide primer pair having a second 5 ′ peptide label. Has the same nucleotide sequence as
Further methods are provided.

In a third embodiment, the invention comprises the following steps: (a) RNA-dependent DNA polymerase and 5 ′ dephosphorylation target-specific primer (ie, a primer specific to the gene whose expression is to be monitored) Is contacted with mRNA from the cell, (b) any DNA: RNA hybrid synthesized in step (a) is contacted with a nuclease to remove single-stranded RNA extensions, and (c) step (b) Followed by a DNA: RNA hybrid molecule and a partially double-stranded phosphorylated second primer (
That is, a first primer that is linked to a primer that is not specific to the target (d) and that is complementary to the target-specific primer used in step (a) (this first primer is the first primer)
And (c) a second primer that is complementary to one strand of the double-stranded phosphorylated second primer (the first primer and the second primer). The product ligated in step (c) is labeled by PCR using (labeled with a distinguishable second label) and (e) the PCR product labeled in step (d) is labeled on a solid support. (F) washing the solid support, and (g) washing the washed support in step (f) with the first label. A method for monitoring gene expression comprising contacting with one or more molecules capable of binding the two labels.

The invention further provides, in a third embodiment, that the nuclease is mung bean (mun)
g-bean) nuclease.

The invention further provides, in a third embodiment, a further method wherein the partially double-stranded phosphorylated second primer is an M13 forward sequencing primer. I do.

The present invention further provides, in a third embodiment, a further method wherein the first label is a peptide label.

The present invention further provides, in a third embodiment, a further method wherein at least one of the one or more molecules in step (e) is an antibody.

The invention further provides, in a third embodiment, a further method wherein at least one of the one or more molecules in step (g) is streptavidin-conjugated horseradish peroxidase.

A fourth embodiment comprises the following steps: (a) each cD is obtained by polymerase chain reaction using oligonucleotide primers having a label specific to each library.
Label the DNA insert from the NA library and (b) the DNA labeled in step (a)
And (c) the DNA hybridized in step (b) is replaced with the first cD
Contacting with multiple immobilized antibodies that do not recognize the label specific to the NA library but can recognize the label specific to each of the other multiple cDNA libraries; and (d) multiple immobilizations Recovering DNA that does not bind to the conjugated antibody;
A cDNA that is present in library A, but not in multiple other cDNA libraries
A method for identifying an A insert is provided.

[0029] The present invention further provides in a fourth embodiment, the hybridized DNA from each of the plurality of other cDNA libraries is in excess as compared to the first cDNA library. Further methods are provided. In addition, additional methods are provided using a 2- to 100-fold excess, a 2.5- to 10-fold excess, and where the excess is a 3-fold excess.

The present invention further provides, in a fourth embodiment, a further method wherein the label specific to each library is a peptide label. In addition, 3
Provided is a method consisting of up to 12 amino acid residues. Furthermore, the present invention provides a method when the label is a thermostable protein label.

The present invention further provides, in a fourth embodiment, a further method wherein the plurality of antibodies in step (c) are immobilized on separate affinity columns, one for each. Furthermore, the present invention provides a method of physically connecting different affinity columns in series in any order.

The present invention further provides, in a fourth embodiment, a further method wherein the column effluent is applied one or more times to separate physically connected affinity columns. Further provided is a method of applying the column effluent three times to separate physically connected affinity columns.

The present invention further provides, in a fourth embodiment, a method for recovering and cloning DNA carried by an antibody specific to a label specific to the first cDNA library.

[0034] In a fifth embodiment, the present invention provides a method comprising the steps of:
A method for identifying a cDNA insert that is present in a cDNA library but not present in a plurality of other cDNA libraries, comprising the following steps: (a) an oligonucleotide having a label specific to each library; By PCR using primers,
(B) hybridizing the DNA labeled in step (a); and (c) hybridizing the DNA hybridized in step (b) with a plurality of other cDNAs.
Contacting with a plurality of immobilized antibodies capable of recognizing a label specific to each of the libraries but not a label specific to the first cDNA library or the second cDNA library; and (d Recovering DNA that does not bind to the plurality of types of immobilized antibodies.

[0035] The present invention further provides, in a fifth embodiment, hybridized DNA from each of the other plurality of cDNA libraries as compared to those from the first cDNA library and the second cDNA library. To provide a method when the amount is excessive. Further, the excess is 2 to 100-fold excess, the excess is 2.5 to 10-fold excess,
And a method is provided where the excess is a three-fold excess.

The present invention further provides, in a fifth embodiment, a method wherein the label specific to each library is a peptide label. Further, the peptide label may be 3 to 12
A method consisting of two amino acid residues is provided. Further provided is a method wherein the label specific to each library is a thermostable protein label.

The present invention further provides, in a fifth embodiment, a method of immobilizing each of the plurality of types of antibodies in step (c) on a separate affinity column.

Further, there is provided a method of connecting the separate affinity columns physically in series in any order.

The present invention further provides, in a fifth embodiment, a method wherein the effluent of the column is applied one or more times to separate physically connected affinity columns. In addition, a method is provided in which the effluent of the column is applied three times to separate physically connected affinity columns.

The present invention further provides, in a fifth embodiment, the DNA recovered in step (d),
Further, the antibody is contacted with an antibody specific to a label specific to the first cDNA library or a label specific to the second cDNA library, and thereby specific to the first cDNA library and the second cDNA library. The present invention provides a method for enriching a specific cDNA fragment.

Further, the present invention provides a method for collecting and cloning concentrated cDNA fragments specific to the first cDNA library and the second cDNA library. In a further method, there is provided a method of isolating enriched cDNA fragments specific to a first cDNA library and a second cDNA library. Further, denatured, coated with the single-stranded binding protein, and the first cDNA library or the second cDNA
The present invention provides a method for performing the above-mentioned separation by contacting an antibody specific to a label specific to the NA library.

In a sixth embodiment of the present invention, there is provided a method for matrix analysis of a plurality of cDNA libraries, comprising the following steps: (a) identifying each of the plurality of libraries with an identifiable label; Labeling the cDNA insert from step (a);
And (c) contacting the cDNA insert hybridized in step (b) with the affinity column capable of binding to the discriminable label; and (d) eluting the affinity column. Providing a method comprising:

The present invention further provides the oligonucleotide according to the sixth embodiment, wherein the discriminating label is a peptide label, and the labeling step has the peptide label attached to its 5 ′ end. Additional methods are provided that include initiating PCR from vector sequences of a cDNA library using primer pairs.

The present invention further provides, in a sixth embodiment, a method in which labeled cDNA fragments from each library hybridize at the same ratio.

The present invention further provides, in a sixth embodiment, a method wherein the affinity column capable of binding to the identifiable label is an antibody affinity column. Further, the present invention provides a method for eluting the antibody affinity column with a pH gradient.

The present invention further provides, in a sixth embodiment, a method for denaturing eluted DNA and separating strands from two different libraries.

In addition, the denatured chains can be singulated by the following steps: (a) coating with a single-stranded binding protein; and (b) contacting with an affinity column capable of binding a discriminating label. A method of releasing is provided.

The method of the present invention may also be used in another embodiment for constructing a subtracted cDNA library. Using the sequences obtained by any of the above methods, the homolog can be obtained from either a library known to share the homolog or a predetermined library unknown about it. The library to be subtracted exists as a purified double-stranded clone. In this example, the ability of E. coli RecA protein to form a stable triple-stranded structure between homologous sequences is used. The triple-stranded structure exists as single-stranded filaments coated with RecA and double-stranded linear and cyclic duplexes. The method of this example comprises the following steps: (a) a sequence identified as a common sequence in different libraries by PCR using a vector-specific primer having a 5 ′ peptide tag,
Amplify and label; (b) denature the tagged sequence; (c) cool (eg, flash freeze) denatured products to avoid regeneration; (d) aliquots of RecA protein and non-hydrolysable Adding ATP (eg, ATPγS); (e) melting the mixture; and (f)
Adding a fixed amount of the library to be subtracted, in the form of a closed circular clone (eg, a plasmid or phagemid).

[0049] The present invention may operate two or more composite population of nucleic acids (e.g., labeling, sorting, isolating and / or screening) for, in general ValiGene ^SM peptide labeled oligonucleotide A method called the VG-PLO ^SM method is provided. Such nucleic acids can be from a variety of sources, and typically can be from cDNA libraries that exhibit different phenotypes. For example, the cDNA library used may be (i) simultaneously present in a given tissue (e.g., normal and cancerous sites of a biopsy specimen), (ii) appear in different tissues belonging to the same or different individuals, (iii) Appear in different cell lines or (iv) 1
It may represent a phenotype that appears in the same cell line with more than one different treatment.

In a general method of the present invention, utilizing the polymerase chain reaction (PCR)
The inserts contained in a given cDNA library are linearized, and an identifiable and identifiable label is added to all inserts from the given cDNA library, resulting in a library (and cell Species, tissues, and organisms). In a preferred embodiment, a PCR reaction is started from the vector sequence of the cDNA library using an oligonucleotide primer to which a peptide label is bound.

Throughout this application, reference will be made to peptide labels and antibodies that bind the labels. One of skill in the art will appreciate that in addition to peptide-antibody combinations, any label may be used in combination with a suitable binding partner. Examples of such labels and binding partners include, but are not limited to, digoxigenin-antidigoxigenin, biotin-streptavidin, ligand (eg, hormone) -receptor, and carbohydrate-lectin combinations.

As used herein, the complexity of a nucleic acid population or mixture of nucleic acids generally refers to the number of distinguishable clones in any given cDNA library or mixture of libraries. Will be understood by those of ordinary skill in the art. The degree of complexity of the nucleic acids analyzed by the method of the invention may vary over a wide range. Generally, there is no upper or lower limit on the degree of complexity of the population or mixture being analyzed. For example, in certain embodiments, the complexity of a population or mixture can be between 10 and 10,000,000. Further, the composite degree may be 100 to 1,000,000. Furthermore, the degree of compounding may be 500 to 500,000. In a preferred embodiment, the complexity of the mixture to be analyzed is about ( + 20%) 150,000. In another preferred embodiment,
A mixture with a complexity of ( + 20%) of 150,000 contains five libraries, each with a complexity of about 30,000. In other specific embodiments, the degree of complexity of the analyzed population is at least 10 ³ , 10 ⁴ , 10 ⁵ , or 10 ⁶ .

The methodology of the present invention uses a library-specific label bound to a primer pair that can recognize the vector sequence of the vector used to construct the library. In this method, all nucleic acid fragments generated by PCR amplification using such primers have the same vector sequence at the 5 ′ and 3 ′ ends, but also contain an identifiable label indicating the library derived therefrom. I have. The following describes a method for selecting a cDNA fragment for isolating a cDNA fragment capable of distinguishing a single cDNA library;
A method for selecting a cDNA fragment for isolating a DNA fragment; a method for selecting a cDNA fragment for isolating a cDNA fragment common to a plurality of libraries but not common to all libraries; and a live library for analysis. A method for selecting a cDNA fragment for isolating a fragment common to only two libraries among all the libraries. In addition, a method of constructing a subtraction library to monitor the phenomenon of gene expression and to isolate full-length transcripts for partial-length sequences such as the Expressed Sequence Tag is described.

5.1 Identification of cDNA Fragments Specific to One of the Multiple cDNA Libraries In this embodiment, inserts from each of the multiple cDNA libraries (eg, A, B, C, D and E) are each Each is linearized and a tag is added by PCR using an identifiable label (eg, a peptide tag containing an epitope) bound to a vector-specific primer. The nucleotide sequence of the vector-specific primer pair may be identical between the libraries. However, each label should be specific to the library of origin. Thus, all fragments generated were (a) identical nucleotide sequences corresponding to the vector-specific primer sequences at their 5 'and 3' ends, and (b) a label indicating the library of origin. Having. The labeled insert is then purified, for example, by exclusion chromatography, to remove any reaction composition containing excess peptide-labeled primer. Next, these purified tagged PCR products are mixed, heat denatured and re-annealed.

The conditions for performing regeneration and hybridization may vary. In a preferred embodiment of the present invention, both of the mixed heat-denatured PCR products are left at 98 ° C. for 10 minutes. The solution is then cooled from 98 ° C to 85 ° C over 5 minutes. Maintain the temperature of the mixture at 85 ° C for 10 minutes, then cool to 65 ° C over 15 minutes. The solution is then maintained at a temperature of 65 ° C. for a further 15 minutes. At this point, the reannealing process is considered complete.

Here, the amount of each PCR reaction product used in the hybridization reaction may also vary. The other libraries are used in excess (as "mops"), as it is desired to isolate cDNA fragments specific to one library. Specifically, if one wishes to isolate fragments that are present in library A but not in libraries B, C, D and E, an excess of B, C, D and E is used.
The amount of excess B, C, D and E used determines the efficiency of the removal. In a preferred embodiment, a three-fold excess of B, C, D and E is used. In another embodiment,
A 2- to 100-fold excess of B, C, D and E is used. In yet another embodiment, a 2.5-10 fold excess of B, C, D and E is used. The role of using excess B, C, D and E is to play the library of interest (library A in this example)
To act as a "mop" to remove homologous cDNA fragments from
There is no limit on the upper limit of excess that can be used. However, a three-fold excess would allow efficient removal of fragments from library A that would hybridize with libraries B, C, D and E without wasting.

Next, an aliquot of the reannealed mixture is passed through, for example, a separate chromatography column to form a binding partner specific to each of tags B, C, D and E (preferably antibody (Ab) ) Column). Methods for producing Ab affinity columns are well known. For example, agarose beads (eg, Seph
arose ^™ or Sepharose CL, Pharmacia) may be activated for Ab binding by use of carbonyldiimidazole or cyanogen bromide. The beads are washed with an aqueous solution of dioxane and incubated with carbonyldiimidazole at room temperature. After incubation, the beads are washed again with dioxane. Next, the purified solution of the desired antibody may be added to the activated beads and mixed overnight at room temperature. Next, the beads are washed with 1 M NaCl and ethanolamine is added. Thereafter, the beads may be directly bound to the antigen or stored.
Many other methods for preparing antibody affinity columns are known to those skilled in the art (see, inter alia, Antibodies-A Laboratory Manual , edited by Harlow, Lane, D., Cold Spring Harbor Laboratory Press, 1988, pp. 519-540). thing).

Many different antibodies can be used in the single and multiple antibody affinity columns used in various embodiments of the present invention. In a preferred embodiment, the label /
An antibody that specifically binds to a short peptide of 6 to 12 amino acids used as a tag is used. However, any length peptide can be used as a "tag" if the selected peptide is known to regenerate spontaneously after thermal denaturation (eg, a thermostable protein). It is. In a preferred embodiment, the antibody releases the retained peptide label when placed in a weakly acidic solution (eg, pH 5.5). Elution of an antibody affinity column based on a pH gradient is another preferred embodiment of the present invention.

[0059] The flow-through is applied to each B, C, D and E column or other solid phase.
Can be applied more than once. Multiple times are preferred. After a few cycles, all or most of the fragments bearing the B, C, D or E tags, either single or double stranded, are captured. In a most preferred embodiment, the effluent is applied to the column three times. The effluent usually contains single-stranded or double-stranded fragments that carry only the A tag.

The temperature at which the antibody affinity column is run may vary. In a preferred embodiment, the temperature is between 4 ° C and 50 ° C. In a most preferred embodiment, the antibody affinity column is maintained at a temperature of 37 ° C. using a water jacket.

Next, the B, C, D, and E columns are washed with a low salt concentration (eg, 50 mM NaCl) buffer (eg, a phosphate buffer). The effluent and the wash are pooled. Next, the pooled effluents and washes from the B, C, D and E columns are passed through a column containing only A-specific antibodies. Next, the captured A-tagged fragment may be eluted, precipitated, and amplified using PCR using unlabeled vector-specific primers. In a preferred embodiment, the A-tagged fragment is amplified using 20 cycles of PCR and cloned for analysis. These cloned fragments are fragments specific to library A (ie, libraries B, C
, D or E) are highly enriched.

[0062] The peptide-labeled fragment may be eluted from the antibody affinity column used in various embodiments of the invention using any method known in the art. In a preferred embodiment, the column changes pH (pH gradient), as described above.
Is eluted. In a most preferred embodiment, the antibody or other solid phase chosen is
The peptide label is released at a slightly acidic pH (eg, 5.5).

Using the same sequence of steps, by changing the “mop”, one can isolate cDNA fragments tagged with B, C, D or E peptides. For example, to isolate a fragment labeled with B, start with an A, C, D and E multiple antibody affinity column and retain all but the B labeled fragment. The effluent and washings of this pooled column are then passed through a column containing only B-specific antibodies. Using the same approach, fragments specific to the C, D and E libraries can be isolated.

In the above example, when isolating the A-specific fragment, the material captured in the first column (multiple antibody column) was all labeled with B, C, D and E labels. Contains fragments. This includes hybrids containing one A-tagged strand. This material is also eluted and further sorted using other embodiments of the invention or as desired by the operator.

5.2 Identification of cDNA Fragments Specific for Two or More of Multiple cDNA Libraries In this embodiment, the methods of the invention are used to share cDNA fragments from one or more other libraries. CD (ie, forming a hybrid with the cDNA)
Isolate the NA fragment. For example, a plurality of cDNA libraries (eg, A, B, C, D
And E), this embodiment allows for the isolation of transcripts common to the A and C libraries (or any other two specified libraries). In this example, for illustrative purposes, the isolation of a cDNA fragment from the A library that forms a hybrid duplex with a cDNA fragment from the C library is described.

In this embodiment, as in section 5.1 above, multiple cDNA libraries (
A, B, C, D and E) were linearized with inserts from each of the above and PCR using discriminating library-specific labels linked to vector-specific primers.
To add a tag. Thus, as described above, all resulting fragments contain at their 5 'and 3' ends the same nucleotide sequence corresponding to the vector-specific primer sequence and an identifiable label indicating the library of origin. Have. Next, the labeled PCR product is purified by exclusion chromatography to remove any reaction composition containing excess labeled primer. Next, the purified labeled PCR
The products are mixed, heat denatured and re-annealed.

In this embodiment, as in the first approach above, the amount of each PCR reaction product used in hybridization may vary. In this embodiment,
If you are interested in isolating the A: C hybrid, use excess libraries B, D and E. The purpose of this excess is to act as a "mop", as in the previous approach. There is no upper limit for this excess B, D and E. However, a three-fold excess effectively removes from both the A and C libraries cDNA fragments that hybridize to any of the fragments from the B, D and E libraries, without waste. Thus, a three-fold excess is a preferred embodiment. In another embodiment, a 2- to 100-fold excess of B, D and E compared to A and C is used. In another embodiment, a 2.5- to 10-fold excess of B, D and E compared to A and C is used. The degree of excess of B, D and E is "
Determine the efficiency of the mop. Although less than a two-fold excess can be used, at the end of this procedure, no significant amount of the A or C fragment will remain that can hybridize with the fragment in B, D or E.

The reannealed mixture is then passed through a chromatography column containing antibodies specific for the labels in all but two of the libraries of interest. In this example, the column contains antibodies to the labels of libraries B, D and E. The effluent of this multiple antibody column may be applied at least once. But,
Multiple times are preferred. This is because each pass increases the proportion of B-, D- or E-labelled fragments retained on the column and thus removed from the effluent. After several cycles, all or most of the fragments bearing the B, D or E labels are removed, and the effluent contains only those fragments bearing the A or C labels. These fragments consist of A: A and C: C duplexes, and may additionally include A: C hybrids (ie, the fragment of interest). These A: C hybrids contain cDNA fragments from the A library that form a hybrid duplex with the fragments from the C library, and are removed by "mops" of the fragments from the B, D, and E libraries. There was no.

This A: C hybrid is the product of interest. Next, the mixture containing the A: A and C: C duplexes and the A: C hybrid is passed through an antibody affinity column having immobilized anti-A labeled antibodies. This column should have at least one
Carry all or most of the fragments carrying the two A-labels. This is A: A
Includes duplex and subject A: C hybrids. The effluent may be applied to the single antibody column one or more times. The amount of A-labeled fragments retained increases with each pass. In a preferred embodiment, the effluent is passed through the column three times.

The substance retained by the column is eluted here, and the trapped substance is recovered.
Let it settle. The recovered material consists of A: A duplex and A: C hybrid. Next, these double-stranded fragments are heat denatured.

Immediately after denaturation, the resulting single strand is rapidly cooled to prevent regeneration. For example, this can be accomplished by rapidly cooling the heat denatured material in a bath of dry ice and methanol. To this frozen mixture is added a single-stranded binding protein (SSB). This protein stabilizes the single-stranded DNA by coating it,
This prevents regeneration. In this example, SSB is added in excess to the denatured single-stranded frozen mixture. The frozen mixture is then warmed so that the SSB enters the solution and comes into contact with the single strand. In a preferred embodiment, the mixture is dry ice /
It is heated from the temperature of the methanol bath to 37 ° C. and left at that temperature for several minutes (for example, 5 to 10 minutes). SSB coats and stabilizes single-stranded DNA and prevents hybrid re-formation and secondary structure formation.

The single-stranded DNA is composed of a fragment derived from the A library, which forms a hybrid with other fragments derived from the A library, and a fragment derived from the C library, which forms a hybrid with the fragment derived from the A library. Consists of The C library fragment present at this stage is limited to those fragments that can hybridize to the A library fragment and are therefore retained on the anti-A antibody column. In addition, these C library fragments did not hybridize to the excess B, D and E fragments used as "mops" and were therefore not removed by them. Thus, these C library fragments are represented in the A library but not in the B, D or E libraries.

In this embodiment, a further step is now used to separate SSB-coated A and C single strands. This step consists of passing the SSB-coated single chain through an antibody affinity column. This column may contain either immobilized anti-A antibody or immobilized anti-C antibody. When an anti-A column is used, the A-labeled fragment is retained by the column and the C-tagged fragment is retained in the effluent and wash. If an anti-C antibody column is used, C-labeled fragments can be retained by and eluted from the column. In any case, the A-labeled fragment and the C-labeled fragment can be recovered. Next, the recovered fragment is extracted to remove SSB, and PCR is amplified. These C-labeled fragments may be cloned for further analysis or may be pooled probes [ie, "subtraction pr.
obe) "to remove their homologues from the original A or C library (see, eg, Section 5.4 below).

In a variation of this embodiment of the invention, more than one library of interest may be specified. For example, when targeting fragments that can hybridize between libraries A, B and C, but do not hybridize with libraries D and E, the first step may be compared to A, B and C And excess D and E PCR
The reaction product is used. In this embodiment, the multiple antibody column includes an anti-D antibody and an anti-E antibody. The excess D and E used becomes a "mop", removing cDNA fragments that hybridize with any fragment in the A, B or C libraries.
The effluent and washings of this multi-antibody column contain A: A, B: B and C: C duplexes, but (if formed) A: B, A: C and B:
Also includes C hybrid. These hybrids are the products of interest in this embodiment. Here, when this substance is passed through an anti-A column, A: B and A: C hybrids are retained. Anti-B antibody columns retain any A: B and B: C hybrids, and antibody columns containing anti-C antibodies retain C: B and C: A hybrids. The substances retained on these three columns can be eluted and isolated by the method used above. This consists of denaturing the double-stranded hybrid, rapidly cooling in a dry ice / methanol bath, followed by warming in contact with excess SSB. Here, the SSB-coated single chain could be isolated by passing it through an antibody column containing a single antibody specific for either the A, B or C label. The fragments eluted from these columns contain the cDNA fragment of interest.

5.3 Matrix Analysis of Multiple Libraries In another embodiment of the present invention, an array or matrix between multiple cDNA libraries constructed and manipulated, in part using variations of the procedure described above. Make a comparison. In this embodiment, any cDNA present in two of the N libraries
Fragments can be isolated (where N is the total number of libraries subjected to matrix analysis). In addition, any cDNA present in X of the N libraries
Fragments can be isolated (where X is the number of libraries in which the isolated specific cDNA fragment can be found). Furthermore, two (or three) of the N libraries
(Or X) of the cDNA fragments. In fact, the cD common to any desired number (X) of any number (N) of libraries analyzed
NA fragments can be isolated and it can be determined whether these fragments are common only within a subset of the N libraries analyzed.

The main advantage of this embodiment is that before starting this comparative analysis, it is not necessary to know which genes (ie, cDNA fragments) may or may not be present in a given cDNA library. Is also a good thing. Further, it is not necessary to know the degree of homology (ie, similarity) between cDNA fragments obtained from a plurality of libraries. Furthermore, prior to beginning the analysis, the library of interest must have similar cD
It is not necessary to know whether the NA insert is shared with any other library. That is, the results of the matrix analysis reveal which cDNA fragments are common (ie, sufficiently similar to hybridize) in any two or more of the libraries of interest. I do.

To illustrate this embodiment, here again, five cDNA libraries (A
, B, C, D and E). First, for each library
The cDNA inserts are separately linearized and labeled by PCR with an identifiable label for each library. Also in this case, a 5'-peptide label is preferable. As described above, a peptide label capable of distinguishing each library was used for 5 ′ of the oligonucleotide primer pair used for performing PCR from the library vector sequence.
Attach to the end.

After the steps of PCR linearization and labeling, the composition of each labeled library is separately purified, for example by exclusion chromatography. In this step, the linearized labeled insert is purified by removing unnecessary reaction components such as excess peptide-labeled primer. This purification can be accomplished by standard methods well known in the art (eg, Qiagen
, Santa Clarita, California).

Next, purified and identifiable labeled cDNA fragments from each of the five libraries were mixed together, heat denatured, and re-annealed. The relative proportions of the materials from each library that are mixed together in this reaction are determined by the practitioner's judgment. This reaction is in excess compared to one or more other libraries.
Substances from one or more libraries may or may not be used. In a preferred embodiment, the labeled cDNA fragments from each library are mixed together in equal proportions.

As in the method of the invention already described, this embodiment provides for labeled cD
A comparative analysis of the cDNA library is performed based on the hybridization and sorting of the NA insert. However, here the analysis utilizes up to four aspects or steps using an affinity column or any solid phase that can bind a particular library label. As described above, any label known in the art suitable for labeling a cDNA strand by PCR can be used. Furthermore, any affinity column or any solid phase known in the art that can bind such labels can be used. In a preferred embodiment, the affinity column is an antibody affinity column capable of binding a peptide label.

The exact number of steps used in this embodiment, and the order in which they are used, is determined in part by the result desired by the practitioner. In this regard, it is not necessary to analyze every product of the comparison array that is made, and it may be stored. For example, if one is trying to isolate a common insert between any two libraries of interest and is not interested in isolating the insert present only in these two libraries, the analysis may be performed in step I All you have to do is II. However, if one wishes to isolate fragments that exist only between libraries within one library group, then step I
, II and III are used. In addition, if one wishes to isolate fragments present only in libraries from different library groups, steps I, II and IV are used. A library "group" is defined as the eluate obtained from the Stage I column, as described further in the section below. The following description illustrates the steps used to achieve the results just described herein. As mentioned above, although antibody affinity columns and peptide labels have been described, other types of solid phases having other types of binding partners for other types of labels can be used.

5.3.1 Step I In step I, the reannealed mixture of labeled cDNA fragments is applied sequentially to a series of antibody affinity columns, each column containing an immobilized antibody capable of recognizing only one of the library labels. Having. Alternatively, the reannealed mixture may be divided into aliquots and applied separately to each column. In this example of five libraries, five columns are used. The order of applying the reannealed mixture to the five separate columns can be in any order. For example, the order is A column, B column, C column, D column and E column, respectively.
It may be a column. In a preferred embodiment, the five columns are physically connected in series. This arrangement has the advantages of flowing the column, minimizing the volume applied to the column, and reducing loss of column effluent and wash. When physically connecting the series of columns in this stage I, the order of the columns in the series is again not important and can be in any order.

Each column of the Stage I series captures or retains a cDNA fragment with one particular label. Therefore, the A column holds all A-labeled fragments, and the B column holds all B-labeled fragments. For example, fragments retained by the A column are labeled A hybrids (ie, A: A, A: B, A: C, A: D
And A: E duplex) and single-stranded A-labeled DNA.

Next, each of the five Stage I columns is eluted separately. If columns AE are physically linked to apply the cDNA mixture and wash, disassemble them before eluting. The material obtained from the column eluate of each step I defines a separate library group as follows. Library group A- from the A column Double-stranded A: A, A: B, A: C, A: D and A: E duplex, and single-stranded A-labeled DNA; Library group B- from the B column Double stranded B: A, B: B, B: C, B: D and B: E duplex, and single stranded B labeled DNA; library group C from C column Double stranded C: A, C: B, C: C, C: D and C: E duplex, and single stranded C labeled DNA; library group D from D column-double stranded D: A, D: B, D: C, D: D And D: E duplex, and single stranded D-labeled DNA; library group E from E column-double stranded E: A, E: B, E: C, E: D and E: E duplex, and one Strand E-labeled DNA;

In this embodiment, the substance of interest is duplex DNA formed from the hybridization of strands from two different libraries. If five libraries are used for the Stage I input mixture, as in this example, twenty different duplexes of interest can be formed. For example, in column A,
The target duplexes of library group A to be captured are A: B, A: C
, A: D and A: E duplex. In column B, the target duplexes of library group B to be captured consist of B: A, B: C, B: D and B: E duplexes, and so on. See FIG. 1, Step I, for a complete listing of the array of products to be produced. Duplexes and any single-stranded DNA having the same label on each strand captured on the column of Stage I are generally not of interest and are therefore not shown in FIG.

5.3.2 Stage II cDNA fragments (ie transcripts) common between any two libraries of interest
Is isolated in step II. Where the eluate from each of the five Stage I columns is:
After being made single-stranded, it is input to the column of Step II. This can be done by any method known in the art. In a preferred embodiment, a single-stranded binding protein (SSB) is used. Thus, the five Stage I columns (group A in FIG. 1)
The eluates from each of ~ E) are separately heat denatured and rapidly cooled (e.g. dry ice / methanol bath). Excess SSB is added, and then each SSB-containing DNA mixture is warmed. In a preferred embodiment, the temperature is raised to 37 ° C, where it is maintained for 10-15 minutes. SSB coats the denatured single strand, preventing regeneration and formation of secondary structure.

Next, the output of each of the five Stage I single antibody columns (here already denatured and stabilized with SSB) is applied to a series of Stage II columns. In a preferred embodiment, each series of Stage II columns is physically linked. Each stage II column contains a single, immobilized antibody specific for one of the distinguishable peptide labels used to label the incoming cDNA library. The columns of the Stage II series may include as many columns as the number (N) of cDNA libraries to be analyzed. In another embodiment, the columns of the Stage II series include N-1 columns, wherein the excluded columns correspond to a library group (ie, for library group A, column A is May be excluded). The immobilized antibody on each stage II column captures a single chain bearing one of the distinguishable peptide labels. Next, the column of each stage II is eluted separately. In this way, an array of 20 groups of single-stranded cDNA fragments is isolated, wherein each of the 20 groups contains fragments that are common (ie, hybridize) between the two libraries. (See FIG. 1, phase II).

For example, each stage II in the A library group (N-1 approaches)
The following cDNA fragments are isolated in single-stranded form after elution of the column: B column-cDNA fragments derived from the B library and also present in the A library; C columns-derived from the C library A cDNA fragment derived from the D column-D library and also present in the A library; and a cDNA fragment derived from the E column-E library and contained in the A library. CDNA fragment that also exists.

As yet another example, the following cDNA fragments are isolated in single-stranded form after eluting the columns of each stage II in the B library group with an N column approach.

B column-B cDNA fragment only; cDNA fragment derived from A column-A library and also present in B library; C column-C cDNA fragment derived from C library and also present in B library cDNA fragment derived from D column-D library and also present in B library; and cDNA fragment derived from E column-E library and also present in B library.

For each series II column, a similar list of isolated cDNA fragments can be constructed, thereby completing the array (see figure, II stage).

Of course, any fragments in the array generated by the results of the column of Step II may be cloned for further analysis desired by the practitioner. Such a fragment can also be used as a "combination probe" to repair the corresponding double-stranded clone from an existing library using, for example, the RecA method described in detail elsewhere herein. These fragments can also be used for the isolation of cDNA present only in two or more libraries of interest, each within a library group or spanning between library groups, as described further below. Stage
Also used as input to III and Stage IV.

5.3.3 Stage III Stage III is used to isolate fragments that are common only between two or more designated members within one library group. For example, stage III allows the isolation of fragments that are only common between libraries A and B, A and C, A and D and A and E within library group A. In addition, stage III allows the isolation of fragments that are only common between libraries B and A, B and C, B and D and B and E within library group B. This pattern is equally applicable to library groups C, D and E.

Step III is a series of steps II in a given library group as described above.
Starting with single-stranded fragments eluted separately from each of the columns. These fragments are first separately amplified by PCR and labeled with an appropriate peptide label. For example, if library group A is to be subjected to stage III analysis, stage II B
The fragment eluted from the column is amplified using the B-specific peptide label, the fragment eluted from column C in step II is amplified using the C-specific peptide label, and so on.

All members belonging to a given library group are similarly amplified and labeled by PCR. The amplified products are then mixed, denatured and re-annealed. In this example (library group A where libraries AE are being analyzed), the reannealed Stage III input mixture is then divided into four aliquots. The number of aliquots required will depend on the number of distinct labels in the mixture. In this example, the A library group was analyzed and PCR
The labels used during the amplification process are B, C, D and E. Stage III columns consist of four series of affinity columns, each series consisting of three single antibody columns. Each of these four series of columns contains antibodies specific for three of the four labels used in the Stage III PCR amplification step. Here, when the A library group is subjected to stage III analysis, the four series of affinity columns include: Series 1-C, D and E antibodies; Series 2-B, D and E antibodies; Series 3-B , C and E antibodies; Series 4-B, C and D antibodies.

Step III series 1 retains any cDNA fragments labeled with C, D and E so that the B-labeled duplex and single strand remain in the effluent. In library group A, these cDNA fragments are present in libraries A and B but not in C, D or E. Therefore, cDNA fragments present only in libraries A and B have been isolated. Similarly, Stage III series 2 passes only C labeled fragments, Stage III series 3 passes only D labeled fragments, and Stage III series 4 passes only E labeled fragments. Here it already exists only in libraries A and C, only in A and D, and only in A and E
Each cDNA fragment has been isolated.

Therefore, usually, in each series of columns, a column having less than the number of labels used in the amplification step is used. In addition, use a sufficient number of series to handle all different combinations of columns.

In another embodiment, the effluent of each of the four series of multiplex antibody columns described herein is then passed through another antibody column. Each of these columns contains one antibody specific for the labeled fragment passed through the columns of the Stage III series. This step serves to concentrate the fragments. If not, those fragments would be difficult to recover from large volumes of effluents and washes. The fragments retained by these four single antibody columns are eluted and collected. This material consists of enriched cDNA fragments that are particularly common only between two specific libraries. In this example, the recovered fragments are from library A in library group A.
And B, A and C, A and D, and A and E.

5.3.4 Stage IV The results from Stage II are further analyzed in Stage IV to ensure that the cDNA fragments common between any two libraries of interest in the array are within the library group “in”. It can be determined whether or not identification is possible between the library groups “between”.
Thus, the Stage IV analysis complements the Stage III analysis by asking essentially the same problem with different input cDNA fragments. For this reason, the implementer:
It is instructive to compare the results of the Stage III analysis with the results of the Stage IV analysis. As in Stage III, input DNA for Stage IV analysis is obtained from the Stage II results. However, the label that binds in the PCR reaction prior to the Stage IV analysis corresponds to the library group label and does not correspond to the original label of the fragment (FIG. 2).
Within the box).

Significantly, therefore, the label that binds by PCR prior to Stage IV analysis of the Stage II results does not correspond to the library of the particular fragment origin. Instead, the label corresponds to the library (ie, library group) in which homologues of the given fragment were found. In this way, an analysis similar to the Stage III analysis can be performed between library groups.

For example, to perform a stage IV analysis between library groups, all fragments derived from the A library and recovered from the columns of the group B in stage II are labeled with a B peptide label ( (See “Tag B” in the frame of FIG. 2). Similarly, stage II
All fragments from the A library, recovered from the columns of the C, D or E groups in the above, are labeled with C, D and E peptide labels, respectively ("Tag C" in the box of FIG. 2 respectively) , "Tag D" and "Tag E").

After PCR amplification and labeling, all of the differently labeled A library fragments were mixed, denatured, and reannealed. The reannealed mixture is then divided into four aliquots, and each aliquot is passed through a multiple antibody affinity column, similar to the columns of Stage III series 1-4. Thus, each multiplex antibody column contains three label-specific antibodies. Where the Stage IV analysis is performed on fragments from the A library, the four series of Stage IV columns include: Series 1-C, D and E antibodies; Series 2-B, D and E antibodies Series 3-B, C and D antibodies; Series 4-B, C and E antibodies.

As in the case of the Stage III analysis, the effluent and washings from each Stage IV series column are then pooled and applied to a column containing one antibody specific for one uncaptured label. Multiply. For example, pool the results of the Stage IV Series 1 column above and pass through the column bearing anti-B. Representative results for the fragments from the A and B libraries are shown in FIG. 4 (see “Results of Step IV”). As described for the results of Step III, the material eluted from each single antibody column in Step IV is common only between the two libraries (ie, not found in other libraries of the analysis) ) Contains concentrated cDNA fragments.

5.3.5 Further Considerations Fragments common to the three (or more) libraries may also be used to manipulate the Stage III and Stage IV series columns to remove the smaller fragments, thereby reducing others. Can be isolated from For example, A exists in A, C and E but B
Alternatively, for stage IV analysis of fragments from library A, which are instructed to isolate fragments not present in D, a stage IV series column containing only B and D antibodies may be run.

5.4 Use of a Method for Constructing a Subtracted Library In another embodiment, the method of the present invention provides a method for constructing a subtracted c
It can be used to construct a DNA library, ie, to remove similar clones from two or more cDNA libraries. The method described here is
Utilizes the ability of E. coli RecA protein to form a stable triple-stranded structure as a recombinant intermediate. RecA catalyzes homologous pairing and strand exchange reactions during homologous recombination in E. coli. During the first step of the reaction, RecA coats the single-stranded DNA and initiates an exchange reaction between the single-stranded and double-stranded DNA homologous regions. A triple-stranded nucleoprotein intermediate is formed, which is surprisingly stable in the absence of ATP (West, 1992, Annu.
Rev. Biochem., 61 : 603-640).

The cDNA fragments of interest, such as those common among the different libraries identified as described above, are amplified by PCR using peptide-tagged vector-specific primers. Thus, an identifiable peptide tag marks a given set of cDNA sequences. The fragments of such a set are simultaneously used as "subtraction probes". The set of subtraction probes is purified by exclusion chromatography followed by PCR and heat denaturation. Next, this cDNA
Quick freeze the mixture (eg, dry ice / methanol bath). An aliquot of the RecA protein is added to the ice-cold pellet along with a non-hydrolysable ATP (eg, ATPγS). The ice-cold pellet slowly thaws. The low temperature and this ATP
The analog prevents regeneration of single-stranded DNA bound to RecA, thereby leaving the subtraction probe single-stranded and coated with RecA.

The library to be subtracted (in the form of purified double-stranded circular DNA) is added to the thawed pellet so that the RecA-coated single strand is present in large excess (20-50 fold). The mixture is heated to 37 ° C. RecA-coated single strands scan double-stranded cDNA libraries for homologous sequences and pair with such sequences. A triple-stranded recombination intermediate is formed, but no strand exchange occurs because there is no hydrolyzable form of ATP. This triple-stranded structure formed from the single-stranded DNA and the homologous double-stranded DNA is labeled with a specific peptide tag bound to the single-stranded DNA. Such triple-stranded labeled structures can now be separated from unlabeled double-stranded cyclic molecules by passing the solution through a peptide tag-specific antibody column. If the RecA-coated single strand is present in a large excess as compared to the plasmid clone, most or all of the clone corresponding to the labeled fragment is removed from the library.

The method of this embodiment does not depend on the original nature of the nucleic acid used to construct the library. Thus, it can be used with DNA libraries made from cDNA or genomic DNA.

In a preferred embodiment, both the single-stranded fragment and the double-stranded library plasmid have the same ends (ie, 5 ′ and 3 ′ ends) in common over at least 10-15 bases and are homologous. The length of the fragment is at least 350 bp. If strong overall homology exists, complete identity between the fragments for RecA is not required to form a stable triple-stranded structure (eg, Rao et al., 1995, Trends In Biologi.
cal Science 20: 109-113). In another preferred embodiment, the length of the cloned insert does not exceed 1-2 kilobases (kb) so that clones that have only strong localized homology with the subtraction probe are not selected.

5.5 Use of the Method in Monitoring Gene Expression In another embodiment of the invention, a method is provided for monitoring a gene expression event. Although oligonucleotides labeled with particular identifiable peptide labels are used, in this embodiment, the target (ie, gene) to be monitored for expression is known. For example, if the purpose is to define the mechanism of action or physiological effect of a particular drug treatment, these targets may belong to an expression cascade. Another use of the method of this embodiment is to monitor gene expression,
To determine a phenotype based on activation or suppression of a metabolic pathway associated with a particular phenotype. The advantage of this method is that it provides a simple and rapid means for sorting, separating and quantifying the product (corresponding to the target to be monitored for expression) based on the peptide label.

Further, the method of this embodiment allows for direct quantification of the amount of target mRNA present. Briefly, a PCR reaction is performed using unamplified cDNA from the first strand synthesis reaction. With a fixed or limited number of PCR cycles (eg, about 5-20 cycles), the products of this reaction are non-genomic and small in size (2 kb or less) present in the reaction.
Is directly proportional to the initial amount of DNA target. The technique of quantitative PCR is well known to those skilled in the art.

The method of this embodiment can be used to generate gene expression events without having to construct a related cDNA library in advance, or starting from a very small biopsy sample that is too small to allow the construction of a new cDNA library. Direct isolation of transcripts as well as isolation of partial length transcripts.

Due to the sensitivity and versatility of this embodiment, it can be used to analyze the response of a particular phenotype to a given stimulus or set of conditions. In addition, this method
It provides a quick and accurate means for directly examining the physiological effects of any form of therapy that affects gene expression (eg, treatment with steroid hormones). this is
As it is done directly by looking at mRNA production, there is no need to wait for a pronounced clinical effect to indicate production of themselves or serological factors. Thus, the method of this embodiment provides quick and direct feedback to make a quick assessment of the estimated effect of the treatment or to allow therapeutic readjustment to optimize outcomes. Can be used to provide.

In this embodiment, total RNA is extracted from tissue samples using standard methodology well known to those skilled in the art. Total RNA is used for hybridization to target-specific probes. Each of these probes consists of a synthetic oligonucleotide labeled at the 5 'end with a particular peptide epitope or tag and at the 3' end with a fluorophore. The single-stranded probe and a sample of total RNA are mixed under conditions that allow hybridization of the probe (if present) to their target mRNA molecule. Following hybridization, the mixture is treated with a single-strand specific DNase to destroy any unhybridized excess probe, or the peptide tag and the Separate from fluorescent label. Here, those skilled in the art understand that other detectable labels can substitute for fluorescent labels. This mixture is then exposed to a solid surface (eg, an ELISA plate) on which the tag-specific antibodies or other binding partners are arranged, thereby identifying the relative position of each target-specific probe. Only probes that hybridize to those targets will emit a fluorescent or other detectable signal at a particular location on the solid surface, and locations on this array will indicate the presence and identity of the target, The intensity indicates the relative amount of each target in the original RNA sample.

This embodiment can also be used to isolate full-length forms of only partial-length transcripts. Total RNA is previously extracted, and an aliquot of the total RNA is used for the synthesis of the first strand of cDNA using a target-specific non-phosphorylated primer (see FIG. 3).

This synthesis uses an RNA-dependent DNA polymerase without RNase-H activity (ie, reverse transcriptase) (eg, Moloney murine leukemia virus reverse transcriptase).
Methods for performing this are well known to those skilled in the art (Sambrook et al., 1989, Molecu
lar Cloning A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Pr
ess). This synthesis results in a target-specific primer at the 5 'end of the DNA strand.
A DNA: RNA hybrid results. Here, any RNA extension product is single-stranded
Treatment with mung-bean nuclease, which cleaves the RNA extension product, can be removed to create blunt ends.

This reaction can be performed under standard conditions for using mung bean nuclease. For example, the DNA: RNA hybrid is a 50 mM sodium acetate (25
° C. pH 5.0 in), 30 mM NaCl, may be suspended in mung bean nuclease buffer consisting 1 mM ZnSO _4. 1.0 units of mung bean nuclease are added per μg of DNA: RNA hybrid and the mixture is incubated at 30 ° C. for 30 minutes. The enzyme can then be inactivated by phenol / chloroform extraction or addition of up to 0.01% SDS. The blunt-ended hybrid can be recovered by alcohol precipitation. Ko
walski, D. et al., (1976) Biochemistry 15, 4457-4463; McCutchan, TF et al. (1984)
Science 225, 626-628.

Here, the sample is purified by standard exclusion chromatography.
After purification, the sample consists of a DNA: RNA hybrid and the remainder of the total RNA species that was originally present. Exclusion chromatography removes small RNA species (eg, tRNA) and excess target-specific primers.

[0119] At this point, performs ligation using DNA ligase from T ₄ bacteriophage. This ligase catalyzes the formation of a phosphodiester bond between the adjacent 3'-hydroxyl terminus and the 5'-phosphate terminus of DNA or RNA, resulting in the formation of three stranded DNA fragments.
The 5 'end is linked to the 5' end of a double-stranded DNA: RNA hybrid molecule. The primer used is a partially double-stranded phosphorylated second primer (ie, a primer that is not target specific), such as the M13 “forward” sequencing primer (see FIG. 4). ).

[0120] Bacteriophage T ₄ DNA ligase, the primers, the DNA: is only rigidly connected to the end phosphorylated RNA hybrids. However, some of the primer molecules are linked to the 3 'end of the RNA strand of the DNA: RNA hybrid. This does not affect the result. This is because the DNA polymerase enzyme in the next step does not use RNA as a template, and cannot use a template in the 3 'direction. Therefore, even if priming is performed at this site, de novo synthesis is performed. This is because no elongation occurs.

[0121] T ₄ DNA ligase purified from E. coli, New England Biolabs (Waverly, Ma
ssachusetts). This reaction was performed in 50 mM Tris-HCl (pH 7.8), 10 mM
mM MgCl _2, 10 mM dithiothreitol, 1 mM ATP, can be carried out by T ₄ DNA ligase buffer in containing 25 [mu] g / ml bovine serum albumin. In a preferred embodiment, the reaction is performed at 16C for 4-16 hours. Engler, MJ and Richardson, C.
C. (1982), The Enzymes (Boyer PD), Vol. 5, p. 3, Academic Press, San Di
ego, CA.

After the ligation reaction, the sample was purified again by exclusion chromatography and
Amplify by

The PCR reaction was performed with a peptide-tagged primer that was complementary to the target-specific primer used previously, and with a partially double-stranded phosphorylated standard primer that was used in the ligation reaction. Both biotinylated primers are used. In a preferred embodiment, the PCR reaction contains 50 ng of yeast RNA per 30 μl of solution. The number of cycles in this PCR reaction can vary. In a preferred embodiment, no more than 20 cycles are used.

In a variation of this embodiment, the sample is prepared immediately after ligation and before PCR.
It may be treated with Nase. This destroys all RNA strands, including the single strand of total RNA and the RNA strand of the DNA: RNA hybrid molecule. The sample is then purified by exclusion chromatography, PCR amplified and labeled as described above. Next, the amplification product is purified by exclusion chromatography to remove any excess primer.

Here, the amount of the product produced by this PCR reaction was determined by enzyme-linked immunosorbent assay (ELI).
It may be determined by a modified method of SA). The purified reaction mixture can be analyzed in microtiter wells coated with an antibody specific for the peptide label attached to the target-specific primer. Horseradish peroxidase conjugated to streptavidin is then added to bind the retained PCR product to the biotin moiety attached to the standard primer. Next, a horseradish perosidase substrate was added,
Reaction products are quantified (eg, Sambrook et al., 1989, Molecular Cloning A Labora
tory Manual , 2nd Edition, Cold Spring Harbor Laboratory Press 18.75) indicates the amount of target mRNA present in the original sample.

In another embodiment of the method, several different targets can be analyzed simultaneously. First
Two or more target-specific primers can be used in the strand synthesis reaction. A separate peptide-labeled primer that is complementary to the target-specific primer and can be considered identical may be used in the PCR reaction. In this embodiment, the involved primers are chosen to be compatible with regard to their melting temperature (Tm) and their tendency for secondary structure formation.

5.6 Selection of input phenotype The input phenotypes represented by the cDNA library used in the method of the present invention can be selected as desired by those skilled in the art. In addition, to make selections on phenotypes, Iris, F. and Pourny, JL
No. 09 / 007,905, filed Jan. 15, 1998, entitled "Method for Identifying Genes Underlying Defined Phenotypes", which may be used. This co-pending application is incorporated herein by reference in its entirety.

5.7 Methods and Products Used with the Present Invention 5.7.1 DNA Amplification The polymerase chain reaction (PCR) is used with the present invention to provide a desired sequence from a source (eg, a tissue sample, genomic or cDNA library). To amplify. Oligonucleotide primers representing a known sequence can be used as primers in PCR. PCR is typically performed using a thermal cycler (eg, Perkin-Elmer).
Cetus) and a thermostable polymerase (eg, Gene Amp ^™ brand Taq polymerase). As a nucleic acid template to be amplified,
Examples include, but are not limited to, mRNA, cDNA or genomic DNA from any species. PCR amplification methods are well known in the art (eg, US Pat. Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Nat ').
l. Acad. Sci. USA 85, 7652-7656; Ochman et al., 1988, Genetics 120, 621-62.
3; see Loh et al., 1989, Science 243, 217-220).

[0129] Any prokaryotic, eukaryotic, or virus can be used as a source of nucleic acid. For example, nucleic acid sequences can be obtained from the following sources: humans, pigs, cows, cats, birds, horses, dogs, insects (eg, Drosophila), invertebrates (eg, C. elegans), plants, and the like. . DNA can be obtained by standard procedures known in the art (eg, Sambrook et al., 1989, Molecular Cloning A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yor
k; Glover (eds.), 1985, DNA Cloning: A Practical Approach, MRL Press Ltd.
, Oxford, UK, Volumes I and II).

5.7.2 Stringency Adjustment Other methods that can be used with the methods of the present invention include nucleic acid hybridization (eg, Northern and Southern blotting) under low, medium, and high stringency conditions. Is mentioned. Methods for adjusting the stringency of hybridization are well known in the art (eg, Sambrook et al.).
, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbo
r See Laboratory Press, Cold Spring Harbor, New York. Ausubel et al.
Ed., Current Protocols in Molecular Biology series of laboratory techniqu
e manuals, 1987-1994 Current Protocols, 1994-1997 John Wiley and Sons, I
See also nc .; in particular, Dyson, NJ, 1991, Immobilization of nucleic acid.
s and hybridization analysis, Essential Molecular Biology: A practical A
pproach, Volume 2, TA Brown, pp. 111-156, IRL Press at Oxford University
Press, Oxford, UK; each of which is incorporated herein by reference in its entirety). The salt concentration, melting temperature, presence or absence of a denaturing agent, and the type (eg, DNA, RNA, PNA) and length of the nucleic acid to be hybridized are determined according to a method known in the art. These are some of the variables that can be considered when adjusting stringency.

Exemplary (but not limiting) conditions of low stringency are as follows (also Shilo and Weinberg, 1981, Proc. Nat'l. A
cad. Sci. USA 78, 6789-6792). For filters containing DNA, 35
% Formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP,
4% in a solution containing 0.1% Ficoll, 1% BSA and 500 μg / ml denatured salmon sperm DNA
Pre-treat at 0 ° C. for 6 hours. Hybridization is performed in the same solution, modified as follows: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml
Use salmon sperm DNA, 10% (wt / vol) dextran sulfate and 5-20 × 10 ⁶ cpm ³² P-labeled probe. Filter at 40 ° C for 18-
Incubate for 20 hours, then wash for 1.5 hours at 55 ° C. in a solution containing 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS. Replace the washing solution with a fresh solution and incubate at 60 ° C for another 1.5 hours. Filters are blot dried and exposed to autoradiography. If necessary, filters are washed a third time at 65-68 ° C and re-exposed to film.

Exemplary (but not limiting) high stringency conditions are as follows. The pre-hybridization of the filter containing DNA was performed using 6 × SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Fico
Pretreat at 65 ° C. for 8 hours to overnight in a buffer containing II, 0.02% BSA and 500 μg / ml denatured salmon sperm DNA. Filter washing is 2xSSC, 0.01%
Perform for 1 hour at 37 ° C. in a solution containing PVP, 0.01% Ficoll and 0.01% BSA. This is followed by a wash in 0.1 × SSC at 50 ° C. for 45 minutes before autoradiography.

5.7.3 Oligonucleotide Analogs Nucleic acids that can be used with the devices of the invention are often oligonucleotides ranging in length from 10 to about 50 nucleotides. In specific embodiments, the oligonucleotide is 10, 15, 20, or 50 nucleotides in length. Oligonucleotides can be DNA, RNA, or chimeric mixtures thereof,
It may be a derivative or modification, or it may be single-stranded, double-stranded or partially double-stranded. Oligonucleotides may be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof. Oligonucleotides may contain additional groups such as biotin, fluorophores or peptides.

The oligonucleotide may comprise at least one modified base moiety, wherein the modified base moiety is 5-fluorouracil, 5-bromouracil, 5-bromouracil,
Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosylkeosin, inosine, N6-isopentenyladenicin, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- Methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylkeosin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine,
Uracil-5-oxyacetic acid (v), pseudouracil, keosin, 2-thiocytosine, 5-
Methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3 -Amino-3-N-2-carboxypropyl) uracil, and
Selected from, but not limited to, the group consisting of 2,6-diaminopurines.

The oligonucleotide is selected from the group consisting of phosphorothioates, phosphorodithioates, phosphoramidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof. (But not limited to) at least one modified phosphate backbone.

The oligonucleotides or derivatives thereof used in conjunction with the methods of the present invention can be prepared using any method known in the art, for example, using an automated DNA synthesizer (eg, Biosearch,
Applied Biosystems). As an example, phosphotrioate oligonucleotides can be prepared using the method of Stein et al. (1988, Nucl.
Acids Res. 16, 3209) and methylphosphonate oligonucleotides can be prepared by using a controlled porous glass polymer support (Sarin et al.,
Nat'l. Acad. Sci. USA 85: 7448-7451). The oligonucleotide may be an α-anomeric oligonucleotide. Alpha-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, where, unlike the normal β-unit, the chains run parallel to each other (Gautier et al., 1987). , Nucl. Acid. Res. 15, 6625-6641).

Oligonucleotides can be prepared by any method known in the art (eg, Applied Biosys
tems 392/394 DNA synthesizer (standard phosphoramidite chemistry). In addition, reagents for synthesis may be obtained from any of a number of vendors.

[0138] A spacer phosphoramidite molecule is used during oligonucleotide synthesis,
For example, portions of the oligonucleotide where base pairing is not desired may be cross-linked, or labels or tags may be located away from the portion of the oligonucleotide that undergoes base pairing. The length of the spacer can be varied by successive additions of the spacer phosphoramidite. The spacer phosphoramidite molecule is 5'- or 3 '
It may be used as a '-oligonucleotide modifier. Such spacers include Spacer Phosphoramidite 9 (ie, 9-O-dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) and Spacer Phosphoramidite 18 (Ie, 18-O-dimethoxytrityl-hexaethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]
-Phosphoramidites) [both are available from Glen Resarch (Sterling, Verginia)].

[0139] Other spacers are available for use in standard oligonucleotide synthesis. For example, Spacer Phosphoramidite C3 and dSpacer Phosphoramidite can be used to destabilize unwanted self-hybridization events within capture oligonucleotides or to prevent false hybridization events between mismatched template / probe complexes. Can be used to stabilize. Such a spacer, when placed at the 3 'end of the oligonucleotide,
Ensure that erroneous extension products are not created when included in the PCR reaction mixture.

Spacer Phosphoramidit, one spacer available from Glen Research
e C3 [i.e., 3-O-dimethoxytrityl-propyl-1-[(2-cyanoethyl)-(N, N
-Diisopropyl)]-phosphoramidite] may be added to replace unknown bases in the oligonucleotide sequence.

[0141] Branched spacers can be used as one method for increasing the incorporation of a label into an oligonucleotide. Also, such branched spacers can be used to increase the signal detectable by hybridization via a multi-branch capture probe or PCR primer. Branched spacers are commercially available, for example, from Glen Research.

[0142] Biotinylated oligonucleotides are well known in the art. Oligonucleotides may be biotinylated using the biotin-NHS ester method. Alternatively, biotin may be conjugated using biotin phosphoramidite during oligonucleotide synthesis (Cocuzza, 1989, Tetrahed. Lett. 30, 6287-6290). Glen Res
One such biotin phosphoramidite available from earch is 1-dimethoxytrityloxy-2- (N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite. This compound also has a branch point that allows for further additions. The branched spacer used in this biotin phosphoramidite is described by Nelson et al. (1992, Nucl. Acids Res. 20, 6253-6259).

Another 5'-biotin phosphoramidite, [1-N- (4,4'-dimethoxytrityl) -biotinyl-6-aminohexyl] -2-cyanoethyl- (N, N-diisopropyl)-
Phosphoramidites can be used to biotinylate oligonucleotides. This compound is sold by Glen Research under license from Zeneca PLC.

Fluorescent dyes may also be incorporated into oligonucleotides using dye-labeled phosphoramidites. Two such labels are 5'-hexachloro-fluorescein phosphoramidite (HEX) and 5'-tetrachloro-fluorescein phosphoramidite (TET), both of which are available from Glen Research.

5.7.4 Preparation of Labeled Oligonucleotides Oligonucleotides can be labeled with a wide variety of labels for use in various embodiments of the present invention. For example, Burdick and Oakes, Diagnostic Kit and Method Using a Solid Phase Capture Means For Dete
European Patent Publication EP0370694 A2 entitled "cting Nucleic Acid"
(May 30, 1990) discloses a method of linking a label to an oligonucleotide.

[0146] Methods of attaching peptides to oligonucleotides are well known to those of skill in the art,
For example, 1) Soukchareun S. et al., Preparation and characterization of antise
nse oligonucleotide-peptide hybrids containing viral fusion peptides, Bi
oconjug. Chem., 1995, 6 (1): 43-53; 2) Tung CH, et al., Preparation of oligonu.
cleotide-peptide conjugates. Bioconjug. Chem., 1991, 2 (6): 464-465; 3) B
ruick RK, et al., Template-directed ligation of peptides to oligonucleotides.
Chem. Biol., 1996, 3 (1): 49-56; 4) Tung CH, et al., Dual-spacificity interac.
tion of HIV-1 TAR RNA with Tat peptide-oligonucleotide conjugates. Bioco
njug. Chem., 1995, 6 (3): 292-295; 5) Robles J. et al., Synthesis and Enzymati.
c Stability of Phosphodiester-Linked Petide-Oligonucleotide Hybrids, Bio
conjug. Chem., 1997, 8 (6): 785-788; and 6) Rajur SB et al., Covalent Prot.
ein-Oligonucleotide Conjugates for Efficient Delivery of Antisense Molec
ules, Bioconjug. Chem., 1997, 8 (6): 935-940.

Oligonucleotides linked to various peptides for use in the methods of the present invention include, for example, Cybergene SA (11 rue Claude Bernard, z1 nord, 35400, Saint Mal
lo, France) and Glen Research (22825 Davis Drive, Sterling, Virginia 2)
0164). Further information from Glen Research is available through their website (www.glenres.com).

One specific method for linking peptides to oligonucleotides recommended by Glen Research is as follows (see also www.glenres.com). The synthetic peptide having an N-terminal lysine residue is linked to a 5'-thiol modified oligonucleotide using a heterobifunctional cross-linking reagent. One such crosslinking reagent is N-maleimido-6-aminocaproyl- (2'-nitro, 4'-sulfonic acid) phenyl ester (mal-sac-HNSA). mal-sac-HNSA sodium salt is available from Bachem Bio
Available from science. Conveniently, reaction of the mal-sac-HNSA crosslinker with an amino group releases a dianion phenolate (ie, 1-hydroxy-2-nitro-4-benzenesulfonic acid). This dianion phenolate is also a yellow chromophore. Due to this chromophore feature, (i) means for quantifying the degree of integrity of the coupling reaction (where the more yellow the intensity, the more complete the coupling reaction); and (ii) Degree of separation of the activated peptide (ie, the peptide that has been cross-linked to mal-sac-HNSA and can be contacted with the 5′-thiol-modified oligonucleotide) from the free cross-linking agent during gel filtration. Support for monitoring the

The specific steps employed when using the mal-sac-HNSA crosslinking agent can be as follows. First, a peptide having an N-terminal lysine is synthesized. Alternatively,
A peptide having an internal lysine may be used. This is because the ε-amino group of lysine is actually more reactive than the α-amino group of lysine. Second, oligonucleotides having a 5'-thiol group are synthesized using methods known in the art.
Third, an excess of mal-sac was added to the peptide in a sodium phosphate buffer (pH 7.1).
-React with HNSA. Fourth, gel filtration columns (eg, NAP-5, Pharmacia, Upps)
ala, Sweden), the peptide-mal-sac conjugate is separated from free crosslinker and the buffer is exchanged for sodium phosphate (pH 6). Fifth, on a gel filtration column, the thiol-modified oligonucleotide is activated, desalted, and the buffer is exchanged for sodium phosphate (pH 6). Sixth, the activated peptide is reacted with a thiol-modified oligonucleotide. Finally, the peptide-oligonucleotide conjugate is purified by ion exchange chromatography (eg, Nu
(cleogen DEAE-500-10 or equivalent). The elution order from this ion exchange column is first free peptide, then peptide-labeled oligonucleotide, and finally free oligonucleotide.

5.7.5 Antibodies and Peptides Antibodies for use with the methods of the invention include any antibodies known in the art. Such antibodies can be used, for example, to engineer a nucleic acid of interest. In this regard, a nucleic acid is an antigen (eg, a protein, peptide or hapten) that is bound to the nucleic acid itself or (covalently or non-covalently) to the nucleic acid.
Can be manipulated with the antibody bound to. In a preferred embodiment, the nucleic acid is engineered with a peptide antigen covalently linked to a PCR primer. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric and humanized antibodies as described below. In addition, single-chain antibodies, Fab fragments and F (ab ') ₂ fragments, as well as fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, and any of the above epitope-binding fragments are also used. be able to.

The polyclonal antibodies that can be used with the present invention are a diverse population of antibody molecules derived from the sera of the immunized animals. Various techniques well known in the art can be used to generate polyclonal antibodies to the antigen of interest. For example,
For the production of polyclonal antibodies, various host animals such as, but not limited to, rabbits, mice, rats, etc. may be immunized by injection of the antigen of interest or a derivative thereof. Various adjuvants can be used to increase the immunogenic response, depending on the species of the host, eg, Freund (complete and incomplete).
, Mineral gels (eg, aluminum hydroxide), surfactants (eg, lysolecithin), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants [eg, BCG (bacille Calmette- Guerin) and Corynebacterium parvum]. Such adjuvants are also well known in the art.

A monoclonal antibody that can be used with the methods of the invention is a homogeneous population of antibodies to a particular antigen. Monoclonal antibodies (mAbs) against the antigen of interest
It can be prepared by using any technique known in the art that results in the production of antibody molecules by continuous cell lines in culture. These include Kohler and Mils
The hybridoma method first described by tein (1975, Nature 256, 495-497), and more recently the human B cell hybridoma method (Kozbor et al., 1983, Immunology).
Today 4, 72) and the EBV-hybridoma method (Cole et al., 1985, Moloclonal Antib
odies and Cancer Therapy , Alan R. Liss, Inc., pp. 77-96),
Not limited to them. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb used in the present invention is in vitro
It can be cultured o or in vivo.

[0153] Monoclonal antibodies that can be used with the methods of the present invention include, but are not limited to, human monoclonal antibodies. Human monoclonal antibodies
It can be made using any of a number of methods known in the art (eg, Teng et al., 19
83, Proc. Nat'l. Acad. Sci. USA . 80, 7308-7312; Kozbor et al., 1983, Immun.
ology Today 4, 72-79; Olsson et al., 1982, Meth. Enzymol . 92, 3-16).

[0154] Chimeric antibodies can be used with the methods of the present invention. A chimeric antibody is a molecule in which each portion is derived from a different animal species, and includes, for example, those having a variable region derived from a mouse mAb and a human immunoglobulin constant region. A variety of techniques are available for making such chimeric antibodies by splicing genes from mouse antibody molecules with appropriate antigen specificity with genes from human antibody molecules with appropriate biological activity (eg, , Morrison et al., 1984, Proc. Nat'l A
cad. Sci. USA 81, 6851-6855; Neuberger et al., 1984, Nature, 312, 604-608.
Takeda et al., 1985, Nature , 314, 452-454) are available.

[0155] Humanized monoclonal antibodies can be used with the methods of the present invention. Briefly, a humanized antibody is an antibody from a non-human species that has one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. Is a molecule. Various techniques have been developed for the production of humanized antibodies (eg, Queen, US Pat.
No., 585,089, which is incorporated herein by reference in its entirety). The variable region of an immunoglobulin light or heavy chain is comprised of a “framework” region that is separated by three hypervariable regions called complementarity determining regions (CDRs). The extent of the framework regions and CDRs has been accurately determined (Kabat et al., 1983,
Sequences of proteins of immunological interest, US Department of Heal
th and Human Service).

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,
No. 778; Bird, 1988, Science 242, 423-426; Huston et al., 1988, Proc. Nat'l Ac.
ad. Sci. USA 85, 5879-5883; and Ward et al., 1989, Nature 334, 544-546).
May be applied to produce a single-chain antibody useful in the device of the present invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region to each other by amino acid binding to obtain a single chain polypeptide.

[0157] Antibody fragments that recognize a particular epitope can be produced by known techniques. For example, such fragments include F (ab ′) that can be produced by pepsin digestion of an antibody molecule.
) ₂ fragments, and Fab fragments that can be produced by reducing the disulfide bonds of the F (ab ') ₂ fragment, including, but not limited to. Alternatively, a Fab expression library can be constructed (Huse et al., 1989, Science , 246, 1275-1281) to quickly and easily identify monoclonal Fab fragments with the desired specificity.

The more general methods of making and using antibodies are suitable for use with the methods of the invention. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manur
al , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (
This is incorporated by reference in its entirety).

The one-letter amino acid code corresponds to the three-letter amino acid code of the sequence listing described below as follows: A, Ala; R, Arg; N, Asn; D, Asp; B, Asx; C, Cys; Q
E, Glu; Z, Glx; G, Gly; H, His; I, Ile; L, Leu; K, Lys;
F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr;
al.

Antibodies suitable for use with the methods of the invention include Affinity Bioreagents,
Inc., 79, rue des Morillons, 75015, Paris, France. 1) Catalog number PA1-047 (affinity purified rabbit IgG). The corresponding peptide recognized by this Ab is KFSREKKAAKT (SEQ ID NO: 1). 2) Catalog number PA1-039 (affinity purified rabbit immunoglobulin).
The corresponding peptide recognized by this Ab is DQKRYHEDIFG (SEQ ID NO: 2). 3) Catalog number PA1-036 (purified rabbit IgG). The corresponding peptide recognized by this Ab is DLKEEKDINNNVKKT (SEQ ID NO: 3). 4) Catalog number PA1-014 (purified rabbit antibody). The corresponding peptide recognized by this Ab is CTGEEDTSE (SEQ ID NO: 4). 5) Catalog number PA3-013 (affinity purified IgG). The corresponding peptide recognized by this Ab is PEETQTQDQPM (SEQ ID NO: 5). 6) Catalog number PA1-815 (rabbit antiserum). The corresponding peptide recognized by this Ab is QKSDQGVEGPGAT (SEQ ID NO: 6). 7) Catalog number PA3-034 (rabbit polyclonal serum IgG). The corresponding peptide recognized by this Ab is DIGQSIKKFSKV (SEQ ID NO: 7). This polyclonal antibody also recognizes QRADSLSSHL (SEQ ID NO: 8).

Further, the antibodies used with the methods of the present invention can be obtained from Medical & Biological Labor
atories Co., Ltd., 440 Arsenal Street, Watertown, Massachusetts 02171, U
It may be obtained from .SA.

[0162] These include the following. 1) Code No. 561 (rabbit IgG from antiserum). The corresponding peptide recognized is
YPYDVPDYA (SEQ ID NO: 9). 2) Code No. 562 (rabbit IgG from antiserum). The corresponding peptide recognized is
EQKLISEEDL (SEQ ID NO: 10). 3) Code number 563 (rabbit IgG from antiserum). The corresponding peptide recognized is
YTDIEMNKLGK (SEQ ID NO: 11).

[0163] The invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein. Because these embodiments are:
It is intended as a description of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description.
Such modifications are also within the scope of the appended claims. Various references are cited throughout this application, the contents of each of which are incorporated by reference in their entirety into this application.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the results obtained in each of Step I, Step II, and Step III of an antibody affinity column used for selecting a labeled cDNA fragment.

FIG. 2 is a schematic diagram of a cDNA fragment containing the input and the resultant of stage IV of the selection process.

FIG. 3 shows an RNA: DNA hybrid produced by cDNA first strand synthesis. The hybrid contains a target-specific primer at the 5 'end of the cDNA strand.

FIG. 4 shows the ligation of a standard primer partially forming an RNA: DNA hybrid, which is partially ligated only to the RNA strand.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 G01N 33/53 M 33/566 33/566 33/577 B 33/577 33/58 A 33 / 58 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, A, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 BA13 BA20 CB01 DA12 DA13 DA14 DA20 DA36 DA37 DA80 FB02 FB03 FB06 FB07 JA20 4B024 AA11 AA20 CA01 CA04 CA09 HA14 4B063 QA01 QA18 QQ53 QR08 QR14 QR32 QR56 QR62 QS03 QS25 QS34 QS36 QX02

Claims (56)

[Claims] 1. A method for selecting a mixture of nucleic acids derived from a plurality of cDNA libraries, comprising the following steps: (a) distinguishing a DNA derived from each of the plurality of cDNA libraries from each library; (B) DNA labeled with the first label in step (a) and DNA labeled with a different label in step (a) And (c) each library is capable of binding to one of the distinguishable labels.
Selecting the DNA contacted in step (b) using at least one kind of molecule. 2. The method according to claim 1, wherein the label capable of identifying each library is a 5 ′ peptide label. 3. The method according to claim 1, wherein the label capable of identifying one library is biotin. 4. The method of claim 1, wherein at least one of said one or more molecules is an antibody. 5. The method of claim 1, wherein the polymerase chain reaction is initiated from a vector sequence common to a plurality of cDNA libraries using an oligonucleotide primer. 6. The following steps: (d) denaturing the hybrid DNA strand obtained in step (b); (e) removing the denatured single strand in (d) to prevent re-annealing of the strand. (F) contacting the single-stranded binding protein coated with the single-stranded binding protein formed in (e) with one of the labels capable of distinguishing each library; Contacting with one or more molecules capable of binding. 7. The method of claim 1, wherein at least one of said one or more molecules is an antibody.
Or the method of 6. 8. A method for comparing cDNA libraries, comprising the steps of: (a) combining DNA from a first cDNA population with a polymerase chain using an oligonucleotide primer having a first 5 ′ peptide label. (B) DNA from the second cDNA population was labeled by the polymerase chain reaction using an oligonucleotide primer having a second 5 'peptide label; (c) labeled in step (a). Contacting the DNA with the DNA labeled in step (b) under conditions that allow hybridization to occur; and (d) DNA having a first 5 ′ peptide label and a second 5 ′ peptide label Is the first
Separating from a DNA having only the 5′-peptide label or the second 5′-peptide label. 9. The method of claim 1, wherein the first population of cDNAs is derived from one or more cells or organisms that have been subjected to the first condition, and the second population of cDNAs has been subjected to the first condition. Absent,
9. The method of claim 8, wherein the method is derived from one or more cells or organisms of the same type. 10. The method of claim 10, wherein the first population of cDNAs is derived from one or more cells or organisms that have been subjected to the first condition, and the second population of cDNAs has been subjected to the second condition. 9. The method of claim 8, wherein the method is derived from one or more cells or organisms of the same species. 11. The method of claim 8, wherein the first and second cDNA populations are from cells or organisms with different phenotypes. 12. The nucleotide sequence of an oligonucleotide primer pair having a first 5 ′ peptide label and the nucleotide sequence of an oligonucleotide primer pair having a second 5 ′ peptide label are identical. The described method. 13. A method for monitoring gene expression, comprising the steps of: (a) contacting a cell-derived mRNA with an RNA-dependent DNA polymerase and a 5 ′ dephosphorylation target-specific primer; (b) Contacting any DNA: RNA hybrid molecule synthesized in step (a) with a nuclease to remove single-stranded RNA extensions; (c) after step (b), the DNA: RNA hybrid molecule (D) ligating the product ligated in step (c) with a first label, the target specific used in step (a) A first primer complementary to the target primer, and a phosphorylated primer forming a double strand used in step (c), labeled with a second label that can be distinguished from the first label Using a second primer complementary to one strand of (E) contacting the product of the polymerase chain reaction labeled in step (d) with one or more molecules capable of binding to the first label immobilized on a solid support. (F) washing the solid support; and (g) contacting the washed support in step (f) with one or more molecules capable of binding a second label. . 14. The method of claim 13, wherein the nuclease is a mung-bean nuclease. 15. The method according to claim 13, wherein the phosphorylated primer partially forming a double strand is a primer for M13 forward sequencing. 16. The method according to claim 13, wherein the first label is a peptide label. 17. The method according to claim 13, wherein the second label is biotin. 18. The method of claim 13, wherein at least one of the one or more molecules of step (e) is an antibody. 19. The method of claim 13, wherein at least one of the one or more molecules of step (g) is streptavidin-conjugated horseradish peroxidase. 20. A plurality of other cDNAs present in the first cDNA library
A method for identifying a cDNA insert that is not present in a library, comprising the following steps: (a) Deriving from each cDNA library by polymerase chain reaction using oligonucleotide primers having a label specific to each library (B) hybridizing the DNA labeled in step (a); (c) labeling the DNA hybridized in step (b) with a label specific to each of a plurality of other cDNA libraries Is contacted with a plurality of types of immobilized antibodies that can recognize, but does not recognize a label specific to the first cDNA library; and (d) recovering DNA that does not bind to the plurality of types of immobilized antibodies; The above method comprising: 21. The method of claim 20, wherein the hybridized DNA from each of the other plurality of cDNA libraries is in excess as compared to the first cDNA library. 22. The method of claim 21, wherein said excess is 2 to 100-fold excess. 23. The method of claim 21, wherein said excess is 2.5 to 10 fold excess. 24. The method of claim 21, wherein said excess is a three-fold excess. 25. The method according to claim 20, wherein the label specific to each library is a peptide label. 26. The method according to claim 25, wherein the labeled peptide comprises 3 to 12 amino acid residues. 27. The method of claim 20, wherein the label specific to each library is a thermostable protein label. 28. The method according to claim 20, wherein each of the plurality of types of antibodies in the step (c) is immobilized on a separate affinity column. 29. The method of claim 28, wherein said separate affinity columns are physically connected in series in any order. 30. The method of claim 29, wherein the effluent of the column is applied one or more times to separate physically connected affinity columns. 31. The method of claim 29, wherein the effluent of the column is applied three times to separate physically connected affinity columns. 32. The method according to claim 20, wherein the DNA recovered in step (d) is further contacted with an antibody specific to a label specific to the first cDNA library. 33. The method according to claim 32, wherein the DNA carried by the antibody specific to the label specific to the first cDNA library is recovered and cloned. 34. A method for identifying a cDNA insert that is present in a first cDNA library and a second cDNA library and is not present in a plurality of other cDNA libraries, comprising the following steps: (a) labeling DNA from each cDNA library by polymerase chain reaction using an oligonucleotide primer having a label specific to each library; (b) hybridizing the DNA labeled in step (a); (c) the DNA hybridized in step (b) can recognize a label specific to each of the other plurality of cDNA libraries, but the first cDNA library or the second cDNA library;
contacting with a plurality of types of immobilized antibodies that do not recognize the label specific to the cDNA library; and (d) recovering DNA that does not bind to the plurality of types of immobilized antibodies. 35. The method according to claim 34, wherein the hybridized DNA derived from each of the other plurality of cDNA libraries is in excess compared to the DNA derived from the first cDNA library and the second cDNA library. the method of. 36. The method of claim 35, wherein said excess is 2 to 100-fold excess. 37. The method of claim 35, wherein said excess is 2.5 to 10 fold excess. 38. The method of claim 35, wherein said excess is a three-fold excess. 39. The method according to claim 34, wherein the label specific to each library is a peptide label. 40. The method according to claim 39, wherein the labeled peptide comprises 3 to 12 amino acid residues. 41. The method of claim 34, wherein the label specific to each library is a thermostable protein label. 42. The method according to claim 34, wherein each of the plurality of antibodies in the step (c) is immobilized on a separate affinity column. 43. The method of claim 42, wherein said separate affinity columns are physically connected in series in any order. 44. The method of claim 43, wherein the effluent of the column is applied one or more times to separate physically connected affinity columns. 45. The method of claim 43, wherein the column effluent is applied three times to separate physically connected affinity columns. 46. The DNA recovered in step (d) is further contacted with an antibody specific to a label specific to the first cDNA library or a label specific to the second cDNA library. 35. The method according to claim 34, wherein cDNA fragments specific to the first and second cDNA libraries are enriched. 47. The method of claim 46, wherein enriched cDNA fragments specific to the first and second cDNA libraries are recovered and cloned. 48. The method of claim 47, wherein enriched cDNA fragments specific to the first cDNA library and the second cDNA library are separated. 49. The separation is denatured, coated with a single-stranded binding protein, and contacted with an antibody specific for a label specific to the first cDNA library or the second cDNA library. 49. The method of claim 48, wherein the method is performed by: 50. A method of matrix analysis of a plurality of cDNA libraries, comprising the steps of: (a) labeling cDNA inserts derived from each of the plurality of cDNA libraries with an identifiable label; (C) hybridizing the cDNA insert labeled in step (a); (c) contacting the cDNA insert hybridized in step (b) with an affinity column capable of binding a discriminable label; and Eluting the affinity column. 51. The labeling step is performed by using an oligonucleotide primer pair having the peptide label attached to the 5 terminus of the primer pair, wherein the labeling step is performed for the cDNA library. 51. The method of claim 50, comprising initiating a polymerase chain reaction from the vector sequence. 52. The method of claim 50, wherein the labeled cDNA fragments from each library hybridize at the same ratio. 53. An affinity column capable of binding to an identifiable label,
The method according to claim 50, which is an antibody affinity column. 54. The method of claim 53, wherein the antibody affinity column is eluted with a pH gradient. 55. The method of claim 50, wherein the eluted DNA is denatured and strands from two different libraries are separated. 56. The denatured strand is isolated by the following steps: (a) coating with a single-stranded binding protein; and (b) contacting with an affinity column capable of binding a distinguishable label. 56. The method of claim 55.