JP2006503558A - Macrocyclic sense molecule array - Google Patents

Abstract

【Task】
[Solution]
A macrocyclic (LC) sense molecule in the array is described. LC sense molecular arrays are combined with cDNA hybridization to detect differences in expression profiles between different cells. LC sense molecules were purified from non-degenerate clones along with recombinant phagemids and aligned on silanized glass slides. By hybridizing a Cy3 or Cy5 labeled cDNA preparation with an LC sense array at 60 ° C., 29 upstream regulated genes and 6 downstream regulated genes were detected in cancerous liver tissue.

Description

  1. TECHNICAL FIELD OF THE INVENTION

  The present invention generally relates to the use of macrocycle (LC) sense molecules as probes in array systems. The present invention also relates to the use of an LC sense molecule library as a probe for the array system. In particular, the present invention relates to a DNA chip technology using an array to which single-stranded LC sense molecules are bound. The invention also relates to a method for producing such an array, an analysis using such an array, a kit comprising such an array, and their use.

  2. General background and prior art:

  Recent developments in DNA microarray technology make it possible to monitor a large number of cellular transcripts in parallel fashion (Schena et al., Science, 270, 467-470 (1995), Delici ( DeRisi) et al., Science, 278, 680-686 (1997), Iyer et al., Science, 283, 83-87 (1999)). Both physiological and pathological changes in cell function are associated with changes in gene expression patterns. For example, the development of malignant tumors is usually associated with overexpression of oncogenes and decreased expression of tumor suppressor genes. Identification of differentially expressed genes is used as a means of recognizing genes involved in disease processes.

  Methods for detecting differentially expressed genes include Northern blot analysis (Alwine et al., Proc. Natl. Acad. Sci., 74, 5350-5354 (1977), S1 nuclease protection (Berk et al., Cell , 12, 721-732 (1977)), differential display (Liang et al., Science, 257, 967-971 (1992)), sequencing of cDNA libraries (Adams et al., Science, 252, 1651) -1656 (1991), Okubo et al., Nature Genet, 2, 173-179 (1992)), continuous analysis of gene expression (SAGE) (Velculescu et al., Science, 270, 484-487 (1995). )), Subtractive hybridization (Hedrick et al., Nature, 308, 149-153 (1984)) and differential expression analysis (RDA) (Hubank et al., Nucleic Acids Res., 22, 5640-5648 ( 1994), Lisitsyn et al., Science, 259, 946-951 (1993)). However, these techniques are limited by the amount of data obtained from a single experiment and are time consuming to achieve.Using cDNA array hybridization, the expression of thousands or tens of thousands of genes can be studied simultaneously. This has been done so far by doting DNA on a nylon membrane and hybridizing with radiolabeled cDNA (Augenlicht et al., Proc. Natl. Acad. Sci., 88, 3286-3289 ( (1991)) Recently, protocols have been introduced that use oligonucleotide micro-arrays with fluorescently labeled probes on cDNA microarrays on glass slides or on so-called gene chips (Schena et al., Science, 270, 467-470 ( 1995), Lockhart et al., Nature, Biotechnol, 14, 1675-1680 (1996)).

  Despite the variety of array technologies being developed, it is still necessary to identify new array devices that meet the requirements of special applications.

  The present invention overcomes the above problems. The present invention provides a method for preparing an LC sense molecule, a library of LC sense molecules, and a method for producing an LC sense array. They are combined with cDNA hybridization to evaluate their usefulness in detecting differences in expression profiles between different cells. Applicants provide arrays that use LC-sense molecules as probe reagents. Certain bacteriophages, such as the M13 bacteriophage, have a single-stranded circular genome and are conventionally used in DNA sequencing analysis as well as mutation studies. For example, M13 phagemid, a plasmid used to construct recombinant bacteriophages, can be engineered to produce large quantities of circular single-stranded genomic DNA containing target-specific sense sequence inserts. This method of producing an LC sense molecule containing a sense DNA insert results in greater resistance to enzymatic degradation associated with its covalently closed structure, higher binding affinity with complementary nucleic acids, higher sequence fidelity, bone It offers many advantages, such as the elimination of difficult target site searches, no denaturation and simple mass production at low cost.

  The present invention relates to a library containing individual LC sense molecules. The LC sense molecule may include a vector sequence and a probe sequence. Here, the probe sequence exists in the sense direction. The vector may be a single-stranded generating phagemid. Further, the LC sense molecule may have a length of about 1,000 to about 20,000 nucleotides. Individual LC sense molecules may be separated from each other or compartmentalized. In particular, the vector may be pSPORT1, pBluescriptIISK (+/−) or KS (+/−), pGEM-f, M13mp, pCR2.1, pGL2 or pβgal. In particular, the vector may also be M13 bacteriophage, f1 bacteriophage, or fd bacteriophage.

  In another aspect, the invention also relates to an array comprising a plurality of individual LC sense molecules stably bound to the surface of a support. The support may comprise a coating layer of aminosilane, poly-L-lysine or aldehyde. Further, the support may be a glass slide, ceramic, inorganic-organic composite, flexible plastic film, silicone, metal or membrane.

In yet another aspect of the invention, the invention relates to a method of making the above array comprising the following steps.
(I) inserting a nucleic acid fragment into a vector that produces a vector in single-stranded form;
(Ii) preparing a bacterial transformant by introducing a vector containing the insert into competent bacterial cells that make the bacterial transformant;
(iii) infecting a transformant with a helper phage that produces an LC sense molecule;
(iv) isolating the LC sense molecule from the culture supernatant of the transformant; and (v) aligning the LC sense molecule on the surface of the support.

  In the method described above, the nucleic acid fragment may be unidirectionally inserted into the vector in all members of the array or library.

In another embodiment, the invention relates to a method for detecting the presence of DNA in a sample in an individual LC sense molecule population in an array comprising the following steps.
(I) labeling DNA in the sample;
(Ii) bringing the sample containing the labeled DNA into contact with the above-mentioned array;
(Iii) hybridizing labeled DNA in the sample with LC sense molecules in the array; and (iv) measuring DNA binding to the LC sense molecules;
Here, the presence of a signal on the array indicates the presence of DNA for the aligned LC sense molecules.

  In the above method, the label may be a streptavidin-alkaline phosphatase conjugate, chemiluminescent or chemiluminescent label. In particular, the label may be Cy3 or Cy5.

In yet another embodiment, the invention relates to a method for detecting the presence of DNA in two or more nucleic acid molecule samples comprising the following steps.
Labeling a first population of DNA obtained from a first sample;
Labeling a second population of DNA from the second sample with a different label;
Contacting a sample containing a first population of labeled DNA with the array described above;
Hybridizing a first population of labeled DNA in the sample with LC sense molecules in the array;
Contacting a sample containing a second population of labeled DNA with the array;
Hybridizing a second population of labeled DNA in the sample with LC sense molecules in the array; and
The binding of labeled DNA to the LC sense molecule is measured, where the presence of a signal on the array indicates the presence of DNA.

  Contacting the array of at least two populations of labeled DNA to the same array at the same time, or the populations may be made to contact the same array sequentially, or the contact may be made on different arrays, Then compare the results.

  In yet another embodiment of the present invention, the present invention relates to a gene expression analysis kit comprising the array and instructions for using the array to detect DNA in a sample.

The gene expression analysis kit further includes (i) a container containing a primer for generating a test nucleic acid;
(Ii) a container containing dNTP and / or rNTP;
(Iii) a container containing a post-DNA synthesis labeling reagent such as a chemically active derivative of a fluorescent dye;
(Iv) a container containing a DNA synthase;
(V) a container containing a buffer medium;
(Vi) a container containing signal generation and detection reagents; and (vii) instructions for use in DNA detection.

The present invention further includes
Cytochrome P450, subfamily IIE (ethanol-inducible) (GenBank accession number J02843);
Transcription elongation factor A (SII) 1;
EST (H. sapiens) weakly similar to KIAA0206 (GenBank accession number AI193075);
1.3 kb mRNA of human skeletal muscle for tropomyosin (GenBank accession number AI7997037);
KIAA0701 protein (GenBank accession number AI7907037);
MRNA for transcription elongation factors S-II and hS-II-T1 (GenBank accession number NM_003195);
Deafness autosomal dominant 5 (GenBank accession no. AF073308);
KIAA1037 protein (GenBank accession number AI383628);
KIAA0375 gene product (GenBank accession number AB002373);
Prefordin 5 (GenBank accession number AA287397);
KIAA0710 gene product (GenBank accession number AB014610);
Paired homeodomain transcription factor 1 (GenBank accession number U70370);
Outer retina membrane protein 1 (GenBank accession number L07894);
EST (GenBank registration number Z39419);
MYC-related zinc finger protein (purine-binding transcription factor) (GenBank accession number M94046);
Ubiquinone conjugate enzyme E2L3 (GenBank accession number AJ000519);
New human genome mapping of chromosome 1 (GenBank accession number AL040438);
Homo sapiens clone 24421 mRNA sequence (GenBank accession number AF070641);
Homo sapiens mRNA; cDNA DKFZp566J2146 (GenBank accession number AL050081);
Chromosome condensation 1 analog (GenBank accession number NM_001268);
KIAA0902 protein (GenBank accession number AB020709);
Tyrosine kinase related protein 9 analog (A6 related protein) (GenBank accession number AI188660);
EST (S. cerevisiae) weakly similar to ORF YOR150w (GenBank accession number AI129433);
Transcription elongation factor B (SIII), polypeptide 2 (GenBank accession number AW327285) and cofactors required for Sp1 transcriptional activity, subunit 9 (GenBank accession number AA665998),
The invention relates to a method for measuring cancerous liver cells comprising detecting upstream regulation compared to normal liver cells of a gene selected from the group consisting of:

The present invention also provides
Transmembrane protease, serine 2 (GenBank accession number U75329);
PAC272L16, a human gene isolated from chromosome 1 (GenBank accession number AL023754), which is similar to a calcium / calmodulin-dependent protein kinase;
CASP2 and RIPK1 domains (GenBank accession number AA811130) containing adapters with death domains;
Ariadne homolog (GenBank accession number AL040708); and NADH dehydrogenase (ubiquinone) flavoprotein 1 (GenBank accession number AW250734),
Compared with normal liver cells of a gene selected from the group consisting of: a method for measuring cancerous liver cells comprising detecting downstream regulation.

  These and other objects of the present invention will be more fully understood from the following description of the invention and the accompanying drawings and claims appended hereto.

  The present invention will become more fully understood from the detailed description set forth below and the accompanying drawings, which are provided for purposes of illustration only. In addition, these do not limit this invention.

  Herein, “a” and “an” are used to refer to both single and multiple subjects.

  The present invention is based on the discovery that macrocyclic phage genomic molecules containing target-specific sense regions are useful as effective probes for complementary target cDNAs, particularly in array sequences. Preferably, the array is a microarray system. Preferably, the microarray system has LC sense molecules spotted densely on the substrate. The system of the present invention can be used at high throughput in high volume array protocols to measure genes involved in various cell physiological processes.

  As used herein, “array” or “array region on a solid support” preferably refers to a one-dimensional or two-dimensional array of individual regions, each formed on the surface of a solid support. Has a limited area.

  As used herein, an “arrayed library” refers to individual single-stranded LC-sense primary recombinant clones (phage, phagemids, phages) mounted in a two-dimensional array of microtiter dishes or plates. Or assembled in a single-stranded genome of another vector). Each primary clone can be identified by the identity of the plate and the position (column and row) of the clone on that plate. Clonal arrayed libraries can be used in many applications, including screening for specific genes or genomic regions of interest as well as physical mapping.

  As used herein, the term “capable of hybridizing under high stringency conditions” means annealing a DNA strand that is complementary to DNA of interest under high stringency conditions. Similarly, “capable of hybridizing under low stringency conditions” refers to annealing a DNA strand that is complementary to the DNA of interest under low stringency conditions. “High stringency conditions” in the annealing process relate to high temperatures and / or low salt content, which, for example, dislikes hydrogen bonding contacts between mismatched base pairs. “Low stringency conditions” refers to lower temperatures and / or higher salt concentrations than high stringency conditions. If there is substantial and almost perfect complementarity between the two strands, as is the case between DNA strands that encode the same protein but differ in sequence due to the degeneracy of the genetic code, such conditions are Anneal two DNA strands. Appropriate stringency conditions that facilitate DNA hybridization, such as washing at 6 × SSC at about 45 ° C. followed by 2 × SSC at 50 ° C. are known to those skilled in the art and are described in Molecular Biology, John Wiley & Sons, NY ( 1989), 6.31-6.3.6 can be found in Current Protocols. For example, the wash step salt concentration may be selected from a low stringency of about 2 × SSC at 50 ° C. to a high stringency of 0.2 × SSC at about 50 ° C. In addition, the temperature of the washing step can be increased from low stringency at room temperature, about 22 ° C., to high stringency conditions at about 75 ° C. Other stringency parameters are described in Maniatis T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring N.Y., (1982), 387-389. See also Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Volume 2, Cold Spring Harbor Laboratory Press, Cold Spring, N.Y., 8.46-8.47 (1989).

  As used herein, “cDNA library” used for a sense probe library linked to a substrate refers to a library composed of LC sense molecules specific for the target messenger RNA.

  As used herein, a “target cDNA library” as used for a target library is any collection of mRNA molecules that are present in a cell or organism and that are converted to cDNA molecules by reverse transcriptase. That said, the library can then be probed for the specific cDNA (and thus mRNA) of interest.

  As used herein, “compartment” refers to a physical description of each member clone of an LC-sense molecule library. The physical depiction may be in the form of a well such as a multi-well plate. Generally, a 96-well plate or a 96-deep hole plate is used. Other physical barriers may be air by individual spotting on a flat sheet, glass or membrane. In this respect, either a macroarray method or a microarray method is used. It is understood to mean that clone members are separated from each other by compartments. Other barriers may be by encapsulating individual clones with membranous material.

  As used herein, an “individual LC sense molecule” as applied to an LC sense molecule forming a microarray is a different LC sense DNA sequence and / or a different of the same or individual LC sense molecule. By array member is meant to be distinguished from other array members based on concentration and / or different mixtures of individual or different concentrations of LC sense molecules. That is, an array of “individual LC sense molecules” includes, as its members, (i) individual LC sense molecules that may have a specific amount in each member, and (ii) different graded concentrations. By means of an LC sense molecule having a given sequence and / or (iii) an array comprising a mixture of different compositions of two or more individual LC sense molecules.

  As used herein, “filamentous phage” is a medium that produces the LC sense molecules of the present invention. Phage or phagemid may be used. In this example, the desired sequence is inserted or cloned into the medium and an LC sense molecule is generated when a single chain is generated by a phage or phagemid. DNA or RNA bacteriophages may be used for this purpose. In particular, filamentous bacteriophages may be used. Filamentous phages such as M13, fd and f1 have a fibrous capsid with a circular ssDNA molecule. These life cycles involve intracellular intermediate dsDNA replicas that are converted to ssDNA molecules prior to encapsulation. This conversion provides a means to prepare ssDNA. Bacteriophage M13 is suitable for use as a cloning vector.

  The phagemid vector also has a filamentous phage f1 Ori region. Examples include the pBluescript (Stratagene, USA), pGEM-f (Promega, USA), M13mp, pCR2.1, pGL2, pβgal and pSPORT vectors and their derivatives. In particular, M13 bacteriophage phagemid vectors such as pBluescriptSK (+/−) may be used. One advantage of using a recombinant viral vector based on M13 bacteriophage is that the vector can accommodate inserts of various sizes. Since the pBluescript SK (+/−) phagemid vector has the f1 (+/−) origin, the target-specific DNA fragment can be inserted in a desired direction, and the sense direction of the inserted DNA fragment is generated.

  Another useful bacteriophage having a single-stranded circular genome and having an icosahedral shape is FX174. However, this cloning vector has limitations on the size of the insert.

  As used herein, “a macrocyclic sense molecule (LC sense molecule or LC sense DNA)” also refers to a “phage genome sense molecule” that is a single-stranded circular DNA molecule, regardless of the origin of the target cDNA. At least one sense region that is substantially complementary to and binds to the target cDNA sequence.

  The LC sense molecule may be synthesized by various methods. Usually, however, it is produced from a filamentous phage system containing M13 and a phagemid. When a macrocyclic nucleic acid molecule is produced from a phage, it is also referred to as a “phage genomic sense compound”.

  In one aspect of the invention, the LC sense molecule is longer than a normal oligonucleotide sequence of about 15-100 nucleotides. LC sense molecules are at least about 3,000 nucleotides long, with DNA molecules consisting mostly of foreign vector sequences. Typically, this range may be from about 1,000 to about 8,000 nucleotides long depending on the size of the insert and the size of the foreign vector sequence. Although nucleotides from about 3,000 to about 7,000 are useful in the present invention, preferred lengths may range from about 3,300 to about 6,000 bases. It will be appreciated that the size of the LC sense molecule may be altered and optimized without undue experimentation, as long as the LC sense molecule binds selectively and specifically to its complementary cDNA.

  It will also be understood that there is no absolute upper or lower limit on the length of the large circular nucleic acid molecule. This is especially true when the vector is used to generate a large circular nucleic acid molecule. In this case, the combination of the size of the vector sequence and the size of the insert sequence encoding at least part of the target gene will control the length of the single stranded nucleic acid produced. That is, in one embodiment, the nucleic acid molecule will be the same length as the vector accommodates.

  Macrocyclic nucleic acid molecules may contain both target-specific sense sequences as well as foreign sequences such as phage sequences. The exogenous sequence may include sense or antisense forms of various other genes. If the vector is used to generate a nucleic acid molecule, the exogenous sequence may be a vector sequence. The length of the target-specific sense region of the macrocyclic nucleic acid molecule may be outside the range of about 100 nucleotides to about 5,000 bases or more. Usually the range may be from about 200 to about 3,000. In particular, the range may be about 400 to about 2,000. In one embodiment, the target specific sense region may encode the entire gene.

  In other embodiments, the LC sense molecule may be generated from the genome of a phage or phagemid as part of its natural life cycle.

  As used herein, a “library” refers to a disordered collection of cloned DNA obtained from a particular organism. Mutual relationships can be established with physical mapping. Such a library comprises about 10 or more individual clones in a set of libraries, preferably 50 or more, preferably 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 1100, 1500, 2000, 2500, 3000, 4000, 5000, 7000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, or 5000 individual LC sense molecules.

As used herein, “microarray” refers to an array having regions where the density of individual regions is at least about 100 / cm ² , preferably at least about 1000 / cm ² . The area of the microarray has a normal size, for example a range of about 10 to 250 μm in diameter, and may be separated by approximately the same distance in the array as the other areas. The microarray may contain a selected set of LC sense molecules. It can be used to test transcriptional expression or profile of expressed genes in a set of cells.

  As used herein, a “probe” as used in the context of an array system is a tethered nucleic acid having a known sequence. In particular, the probe is connected to a substrate such as a glass slide.

  As used herein, the term “specifically binds” refers to a non-random binding reaction between two molecules, eg, between an LC sense molecule and its complementary sequence.

  As used herein, “substantially complementary” refers to about 80%, 85%, 90%, 96%, 97%, 98%, 99% or 100% similarity to other nucleic acid sequences. Means a nucleic acid sequence having In general, absolute complementarity will not be necessary for specific binding to occur between two nucleic acid molecules. Stable duplex formation depends on the sequence and length of the hybridizing LC sense molecule and the degree of complementarity between the LC sense molecule and the target sequence, thus forming a stable duplex with the target cDNA. LC sense molecules with sufficient complementarity are considered to have suitable specificity for binding between two nucleic acid molecules.

  As used herein, “target” or “targeting” in the context of an array system refers to a free nucleic acid transcript whose identity or amount is sought to be detected using an LC sense probe or Refers to cDNA and specifically refers to the individual gene from which the LC sense molecule is made. In one context, “targeting” means to bind or bind an LC sense molecule to an exogenously expressed transcript or cDNA thereof. The target nucleic acid sequence may be selected without restriction from any gene, in particular genes associated with various malignancies including genes associated with cancer, and immune diseases, infectious diseases, metabolic diseases and genetics It may be selected from genes associated with the onset and progression of various diseases, such as diseases or other diseases caused by abnormal gene expression.

  As used herein, a “unidirectional” or “random gene unidirectional” sense library indicates that the orientation of the inserted gene for each member clone in the library is unidirectional. The term “random” is meant to refer to a library containing genes of unproven sequences.

  As used herein, a “unidirectional subtracted library” is a selective enrichment of genes expressed or overexpressed in a specific tissue or cell line of interest compared to a control tissue or cell line. A library.

  As used herein, “single gene” sense library refers to a collection of sequence-verified nucleic acid fragments optionally inserted into a sense nucleic acid generating vector.

  Macrocycle (LC) sense molecule

  The present invention provides LC sense compounds having great stability and specific activity against nucleases, and methods for producing LC sense compounds by using recombinant bacteriophage with a single-stranded circular genome. The present invention also provides an LC sense DNA library as probe DNA for preparing an array. LC sense molecules that are specific for multiple genes may be produced simultaneously in small or large amounts from bacterial cultures containing recombinant bacteriophages. In an exemplary embodiment of the invention, 1,152 different LC sense samples were obtained in small quantities from 3 ml of culture supernatant and spotted on the surface of a silanized glass slide. In general, 1 to 3 μg of LC sense DNA was obtained from 1 ml of the culture supernatant.

  In another exemplary embodiment of the present invention, Applicants designed a semi-automatic device primarily comprising a purification column, a dispenser and a vacuum manifold for producing LC sense DNA libraries in large quantities. Using this device and its ability to collect 100 ml of culture supernatant, ˜200 μg of LC sense molecule was obtained. The production scale will be expanded to liter units by using a jar fermenter system for cultivation.

  Furthermore, in one embodiment of the present invention, by using the phage genome sense method of the present invention, the efficiency of the array system for high throughput detection of gene expression uses oligonucleotide probes or PCR amplified larger nucleic acid probes. It is superior to conventional methods. That is, the LC sense molecule may be used as a probe in settings or devices where hybridization to its complementary DNA is desired.

  Various methods are currently available for creating an array of DNA probes. The LC sense molecule may be used in such a system as a probe bound to the membrane, such as in a nucleic acid molecule array. One method for creating an array of DNA on a porous membrane is the “dot blot” method. In this method, a plurality of vacuum manifolds, for example, 96 DNA aqueous samples are transferred from a 3 mm diameter hole to a porous membrane. A common variation of this technique is the “slot blot” method, which has an oval shape with a large hole. The DNA is immobilized on the porous membrane by baking the membrane or exposing it to UV radiation. This is a manual procedure that helps to create one array at a time and is usually limited to 96 samples per array.

A more efficient technique used to create an array of genome fragments is to immerse in a microtiter plate hole, eg 96 holes, to transfer the array of samples to a substrate such as a porous membrane. Use a pin array. One array includes pins designed to spot the film in a zigzag pattern to create an array of 9216 spots in an area of 22 × 22 cm ² .

  In recent years, array systems have (i) a large number of micro-sized analytical regions separated by a distance of 50-200 microns or less, and (ii) well-defined amounts of LC bound to each region of the array, typically LC in the picomolar range. Invented for mass production of microarrays featuring sense molecules (US Pat. No. 5,807,522, which specifically relates to microarray systems, the entirety of which is incorporated herein by reference) ).

  According to one aspect of the present invention, the LC sense compound of the present invention comprises 1) preparing a cDNA fragment containing the target nucleotide sequence; 2) cloning the cDNA fragment into a phage vector capable of producing the LC sense compound. May be prepared by generating a single-stranded circular phage genome containing a large amount of the target sense sequence. Such a library of LC sense molecules may be created.

  That is, it is understood that in other aspects of the invention, the LC sense compound may comprise either a fragment of the target sequence or the entire gene sequence. In addition, several target specific sense sequences for multiple different genes may be inserted into a single single-stranded phage genome. Consequently, the LC sense molecule may include one or more regions of the target specific sense sequence.

  The LC sense compound has strong replication fidelity because this compound is replicated by DNA polymerase in bacterial cells. Since DNA polymerase has proofreading capabilities, the fidelity of LC-sense compounds is greater than chemically synthesized oligonucleotides. Furthermore, the LC sense compounds of the present invention are made cheaper than chemically synthesized oligonucleotides or amplified DNA fragments.

  In other aspects of the invention, it is understood that each compartment in the array contains only LC-sense molecules. In other embodiments, the compartment contains both the LC sense molecule and its complementary counterpart, such as when a double-stranded phagemid is denatured to produce an LC sense molecule strand and its complementary single-stranded counterpart. Also good. Preferably, more than 1 to 1 ratio of LC sense molecules may be present in the region of the array as compared to the complementary single-stranded corresponding DNA. More preferably, the composition in the region of the array comprises at least 95% LC sense molecules. Even more preferably, the composition comprises LC sense molecules that are produced as single stranded DNA during the life cycle of the phage containing insert when only single stranded LC sense molecules are present in the region of the array.

  High-throughput microarray system

  Mass expression profiling with arrays has emerged as a major technique in the systematic analysis of cell physiology (Young et al., Cell, 102, 9-15 (2000)). These arrays are now gene discovery (Kati et al., J. Pathol., 193, 73-79 (2001)), disease diagnosis (Alizadeh et al., Nature, 403, 503-511 (2000)), Drug discovery (Leming et al., J. Chem. Inf. Comput. Sci., 40, 367-379 (2000)), toxicology research (Nuwaysir et al., Molecular Carcinogenesis, 24, 153-159 (1999) )) And the like. Techniques for producing microarrays include the technique of forming probe oligonucleotides (usually 15-100 nucleotides) directly on a glass surface (Lipshutz et al., Biotechniques., 19,442-447 (1995), Lipschutz. (Lipshutz et al., Nat. Genet., 21, 20-24 (1999), Singh-Gasson et al., Nat. Biotechnol., 17, 974-978 (1999)), or a cDNA clone set or cDNA Techniques such as plotting substrates with PCR products amplified from the library (Duggan et al., Nat. Genet., 21, 10-14 (1999)) are utilized. However, the production of arrays by oligonucleotides or PCR products has several disadvantages. For example, the preparation of tens of thousands of degenerate oligonucleotides involves multiple time-consuming steps such as sequence information, high production costs and sought target sequences for each gene, synthesis, desalting, column purification, concentration, and denaturation. I need. On the other hand, the production of arrays using PCR products requires plasmid purification, cDNA amplification with Taq polymerase, and costly DNA purification steps. Here we devised an array using LC-sense molecules. Its use has been demonstrated as a binding reagent probe for studying gene expression profiles.

  A plasmid for the construction of recombinant bacteriophage, M13 phagemid, has been incorporated to produce large quantities of single-stranded genomic DNA containing the sense sequence because of its f1 origin. LC sense molecules can be produced in large quantities from competent cell bacterial cultures along with recombinant M13 phagemids by co-infection with helper bacteriophages.

  In this regard, the ability to produce large quantities of LC sense molecules for use in the array provides the advantage of reducing the price of the array. On the other hand, the lack of the ability to easily produce large amounts of oligonucleotides and PCR-produced large cDNA products has been an obstacle to obtaining an inexpensive array chip.

  The present invention also provides a high throughput system for functional genomes using the LC sense molecule library described above. The functional genomic system of the present invention may be used to study gene function quickly and in large quantities. That is, the LC sense library may be used to determine the interrelationship between different gene products.

  Various specific array types, including LC sense molecules, are provided by the present invention to identify genes differentially expressed in various animal, plant and microbial cells or tissues. These array types include: developmental arrays; cancer arrays; apoptotic arrays; oncogene and tumor suppressor arrays; cell cycle gene arrays; cytokines and cytokine receptor arrays; growth factors and growth factor receptors. Array; nerve array and the like, but not limited to.

  The arrays of the invention can be used inter alia for differential gene expression analysis. For example, an array may include: (a) a disease state, such as neoplastic or normal; (b) different tissue types; (c) developmental stage; (d) response to external or internal stimuli; (e) response to treatment, etc. It may be used for genetic expression analysis. This array is also useful for extensive scale expression screening for drug discovery and research. Furthermore, by studying the effects of activators on specific cell types in gene expression, information such as drug toxicity, carcinogenicity, environmental monitoring, etc. can be obtained and analyzed.

In one aspect, the invention includes a surface substrate having a microarray of at least 10 ³ individual LC sense molecules on a surface area of about 1 cm ² or less. Each individual LC sense molecule is (i) located in a spaced position in the array, (ii) has a length of at least about 3,000 bases, and (iii) about 0.1 femtomole to 100 Present in a predetermined amount of nanomolar.

  In one embodiment, the surface may be a glass slide surface coated with a polycationic polymer such as polylysine and is not conjugated to a coating layer, without being limited to a specific substrate or a specific array system. It may include an array of individual LC sense molecules that are bound and electrostatically bound. Here, the individual LC sense molecules are arranged at predetermined positions apart from the surface array.

  A method for detecting each differential expression of a plurality of genes in a first cell type in relation to the expression of the same gene in a second cell type also forms part of the present invention. To perform this method, fluorescently labeled cDNA is first produced from mRNA isolated from two cell types. Here, the cDNAs obtained from the first and second cell types are labeled with first and second different fluorescent reporters.

  A labeled cDNA mixture obtained from two cell types is an LC sense molecule that expresses a plurality of known genes derived from the two cell types under conditions that hybridize the cDNA to LC sense molecules of complementary sequence in the array. Added to the array. This array is then examined by fluorescence under fluorescence excitation conditions. Where (i) LC sense molecules in the array that preferentially hybridize to cDNAs derived from one of the first or second cell types exhibit individual first or second fluorescent emission colors, respectively. And (ii) the polynucleotides in the array that hybridize to a substantially equal number of cDNAs derived from the first and second cells each exhibit their respective combined fluorescent emission color. The relative expression of the known genes of the two cell types can then be measured by the observed fluorescent color of each spot.

  An exemplary mass functional genomics protocol is as follows, and it is understood that the specific embodiments and examples do not limit the invention in any way.

(1) constructing a cDNA library using a recombinant bacteriophage vector having a single-stranded genome;

(2) identifying and selecting cDNA clones with insert size;
(The insert size may be at least 100, 200, 300, 400 bases, preferably at least 500 bases to at least about 2,000, 3,000, 4,000 or 5,000 bases or more. (It may be isolated using a scale plasmid.)

(3) amplify selected clones and construct LC sense library;
(Selected phagemid transformants are infected with helper bacteriophage. Single-stranded phage genomic sense compounds are subsequently collected from the culture supernatant.)

(4) Distribute individual LC sense molecules onto a substrate such as glass, membrane or filter in the array. (The dispensing or spotting process may be performed manually or automatically using a spot machine.)

  The cell from which the target cDNA is obtained can be a cell of interest such as a normal cell or various types such as liver cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, rectal cancer, colon cancer, prostate cancer, thyroid cancer, and skin cancer. You may choose from cancer and mast cells, hair follicles, autoimmune diseases, and cells with metabolic disorders.

  A library of LC-sense molecules can be obtained by inserting a population of cDNA inserts randomly and unidirectionally with a modified shotgun, or by preparing a special, non-degenerate library of clones of interest. The sequences may be identified individually and the insert in the phage vector cloned. It will be appreciated that the source of the random gene unidirectional LC sense library or single gene unidirectional LC sense library or host cell to be tested need not be human. According to the principles of the present invention, organisms of any origin such as, but not limited to, humans, plants and fungi are used. The host cell can also be any organism as long as the LC-sense compound can penetrate the cell membrane or cell wall.

  To evaluate the function of the LC sense molecule as an array binder, we first transformed 1,152 non-degenerate clones of recombinant pSPORT phagemid into E. coli competent cells using the helper bacteriophage M13K07. . The LC sense molecules of each clone were then purified from the culture supernatant and concentrated in large quantities to 0.2-0.5 mg / ml. In this way we obtained 1,152 samples. As an array substrate for the binder, poly-L-lysine or aminosilane was coated on the glass surface to enhance nucleic acid immobilization (Schena et al., Proc. Natl. Acad. Sci., USA, 93, 10614-19 (1996)). After confirming the nature and amount of LC-sense molecules by agarose gel electrophoresis, LC-sense arrays were prepared by spotting these molecules on silanized glass slides using a microarray. After confirming the properties of poly (A +) RNA purified from normal and cancerous liver tissue, labeled cDNA probes were mixed and hybridized with an LC sense array containing 1,152 non-degenerate samples.

  RNA obtained from normal liver cells and cancerous liver tissue was reverse transcribed into cDNA in the presence of nucleotides labeled with radiolabeled or fluorescent tags. Cy3-dNTP and Cy5-dNTP dyes are the most commonly used phosphors as labeling agents. Incubating the target with conventional microarrays containing oligonucleotides or amplified cDNA products was usually performed at 45 ° C. or 65 ° C. in aqueous hybridization buffer, respectively. However, we optimized the hybridization temperature to 60 ° C. based on measuring the melting point of single-chain LC molecules. These differences in the optimal temperature for hybridization reflect structural differences between the LC-sense molecular probe and conventionally used oligonucleotides or PCR products. Following hybridization, the LC sense array was washed repeatedly to remove unbound and non-specific signals. Scanning analysis by software showed that 29 out of 1,152 genes were up-regulated and 6 genes were down-regulated in cancerous liver tissue compared to normal tissue. From these results, we confirmed that the LC sense molecule works well as a probe on the glass slide array.

  LC sense arrays offer several advantages in the use of an array system used to study gene expression profiles. First, LC sense molecules can be produced in large quantities from bacterial transformants such as E. coli with speed, accuracy and cost effectiveness. Secondly, phagemid vectors can accommodate sense inserts of various sizes. Because of its long sequence, binding specificity can be significantly increased. Third, creating an array with LC-sense molecules does not require time spent investigating target binding sequences. Fourth, LC sense molecules have strong replication fidelity because the molecules are replicated by DNA polymerase in bacterial cells. Because DNA polymerase has proofreading capabilities, the fidelity of LC sense molecules is greater than chemically synthesized oligonucleotides or cDNA amplified in vitro. Fifth, LC sense molecules can be made cheaper than chemically synthesized oligonucleotides or amplified PCR products. Finally, using vector-based techniques, the construction of an LC-sense molecule library with a large number of individual clones may be accomplished easily and quickly. Libraries specific for a particular disease can be readily constructed from diseased cells or abnormal cells or tissues. From these libraries, we can produce LC sense molecules in large quantities and find a panel of disease-related genes including genes with unknown functions. Otherwise, diverse expression profiles of various diseases are described herein from libraries constructed with little or no degeneracy among members of the full panel of human genes or genes of other organisms. Can be achieved. In a further step, a combination of inhibitory subtractive hybridization (SSH) and cDNA array hybridization methods may be used to discover anticancer agents more efficiently (Kati et al., J. Pathol., 193 73-79 (2001) A method for creating a unidirectional library of random genes overexpressed in specific cells or tissues using a phagemid vector with f1 origin in the construction of a subtracted cDNA library Gene expression profiles obtained from LC sense arrays have been confirmed by other methods including real time PCR and northern blotting.

  In one general embodiment, the surface is relatively hydrophilic, i.e., a wettable surface, such as a surface having an unmodified, binding or covalently attached charge group. One such surface described below is a glass surface having an absorbent layer of a polycationic polymer such as poly-L-lysine.

  In other embodiments, the surface has, or is formed to have, a relatively hydrophobic property, i.e., the property of beading an aqueous medium deposited on the surface. As with various lubricants or other hydrophobic films applied to glass and support surfaces, various known hydrophobic polymers such as polystyrene, polypropylene, or polyethylene have desirable hydrophobic properties.

  The slide may be coated by placing a uniform film of a polycationic polymer, such as poly-L-lysine, on the slide surface and drying the film to form a dry film. The amount of polycationic polymer added need only be sufficient to form at least a monolayer of polymer on the glass surface. The polymer film may be bonded to the surface by electrostatic bonding between negative silyl-OH groups on the surface and charged amino groups in the polymer. Poly-L-lysine coated glass slides may be purchased from, for example, Sigma Chemical Co. (St. Louis, Mo).

  To form a microarray, a defined amount of individual LC sense molecules are deposited on a polymer-coated slide. According to an important aspect of the substrate, when the aqueous DNA sample is applied to the substrate under conditions that allow the reporter labeled cDNA in the sample to hybridize with the LC sense DNA probes in the substrate array, the attached LC sense molecules It will be in the state couple | bonded with the coating slide surface by the noncovalent bond.

In a preferred embodiment, each microarray comprises at least 10 ³ individual LC sense molecules per surface area of about 1 cm ² or less. The microarray may include at least about 400 regions, or 2.5 × 10 ³ regions / cm ² in an area of about 16 mm ² . In a preferred embodiment, in the case of polynucleotides, the LC sense molecules in each microarray region may be present in a defined amount between about 0.1 femtomole and 100 nonamol.

  Also, in a preferred embodiment, the polynucleotide has a length of at least about 3000 bp, ie, is substantially longer than oligonucleotides that can be formed into a high density array by various in situ synthetic schemes.

  Sign

Suitable enzymes include, for example, those consisting of oxidases that catalyze the production of hydrogen peroxide by reacting with a substrate. Glucose oxidase is particularly preferred because it has good stability and its substrate (glucose) is readily available. The activity of the oxidase label may be analyzed by measuring the concentration of hydrogen peroxide generated by the enzyme labeled antibody / substrate reaction. Besides enzymes, other suitable labels iodine ^{(125 I, 121 I),} carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ⁽³ H), indium ⁽¹¹² an In) and technetium ^(99m Tc) And isotopes, and fluorescent labels such as fluorescein and rhodamine, and biotin.

Examples of suitable radioisotope labels include ³ H, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd and the like. Examples of suitable non-radioactive isotope labels include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Tr and ⁵⁶ Fe.

Examples of suitable fluorescent labels include ¹⁵² Eu label, fluorescein label, isothiocyanate label, rhodamine label, phycoerythrin label, phycocyanin label, allophycocyanin label, o-faraldehyde label, fluorescamine label, cyanine (Cy3 ^™ ) and Indocarbocyanine (Cy5 ^™ ) can be mentioned.

  Examples of chemiluminescent labels include luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels and aequorin labels.

  Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn and iron. Diuterium may also be used. Other contrast agents are also present in EPR, PET or other imaging mechanisms. These are known to those skilled in the art.

kit

  The invention also includes a kit for analyzing a sample for the presence of cDNA in the sample. In a typical embodiment, the kit includes a substrate on which an array of LC sense molecules is present in one or more containers. In a specific embodiment, the kit of the present invention may contain a test nucleic acid that reacts specifically with a reagent, NTP, enzyme, column and array. Preferably, the kit of the present invention may further contain a nucleic acid that reacts or does not react with the microarray. The kit further includes instructions for use and a label.

  The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. The following examples are provided to illustrate the invention and are not intended to limit the invention.

  LC sense array

  Recombinant pSPORT phagemid was infected with helper bacteriophage M13K07 (NEB Nucleic Acids, USA) and incubated overnight at 37 ° C. on LB agar plates containing ampicillin (50 μg / ml). Gold, Stratagene, USA). Fully isolated transformants were added to each of 96 deep well plates containing 2 × YT liquid medium (tryptone 16 g, yeast extract 10 g, NaCl 10 g / 1000 ml) containing 50 μg / ml ampicillin and 70 μg / ml kanamycin. The cells were seeded in the wells and cultured at 37 ° C. for 14 hours with vigorous stirring. This incubation was performed three times for each clone to maximize the yield of LC sense molecules with a single purification. For the production of a small amount of LC sense molecule, 3 ml of culture supernatant was added together with 1/5 volume of 20% polyethylene glycol (PEG 8000) and 2.5 M NaCl and transferred onto the QIAprep 96M13 kit (Qiagen, German). The purification step was performed using a QIAVAC vacuum manifold (Qiagen, German) according to the manufacturer's instructions. The prepared LC sense molecule was placed on a 1% agarose gel to test its quantity and quality. The eluate was then dried, redissolved in 10 μl of 3 × SSC, adjusted to the concentration of LC sense molecules, and silanized glass slides (CMT-GAPS) using an OmniGrid microarray (GeneMachines, Inc., USA). , Corning, USA). Each slide was cross-linked by irradiation with 300 mJ of short wave UV (Stratalinker, Stratagene, USA) and stored in a dryer until use.

  Production of LC sense molecule of rat TNF-α

RatTNF-α cDNA was cloned into the multiple cloning site of the phagemid vector, pBluescript (pBS) -KS (+). Production of recombinant M13 phage was performed by infecting bacterial cells already transformed with pBS KS (+) phagemid with M13K07 helper phage. The phagemid f1 replication origin was used to generate an LC sense molecule for the target gene. 20% polyethylene glycol (PEG 8000) was added to the supernatant of an overnight culture of cells infected with helper phage. The bacteriophage precipitate was resuspended in TE (pH 8.0) and the phage genomic DNA was isolated by fail extraction and ethanol precipitation. For purification of LC sense molecules obtained from helper bacteriophage and residual genomic DNA of host bacterial cells, 0.8% low melting point (LMP) agarose gel is used for small-scale purification, or gel filtration column chromatography ( 1.0 × 50 cm). The gel filtration column resin is a very fine Sephacryl ^™ S-1000 (molecular weight cut-off: 20,000 bp) (Amersham Pharmacia Biotech AB, Sweden), 50 mM Tris packed and containing 0.2 M NaCl (pH 8.3). -Equilibrated with HCl buffer. The initial amount of LC molecules was adjusted to 5% gel hole volume and DNA elution was performed with the same buffer used in resin equilibration (flow rate: 0.3 ml / min). Samples were UV scanned at 260/280 nm using a dual UV detection system and samples were collected every 5 minutes during elution. Sample fractions were washed, precipitated with 70% cold ethanol and resuspended in ultrapure water and PBS (phosphate buffered saline) diluted for the next experiment. Purified LC molecules were tested for their quantity and purity on a 1% agarose gel.

_Tm analysis

Thermal denaturation of double-stranded plasmid DNA (pBS) -KS (+) phagemid containing a single-stranded LC molecule of rat TNF-α and a TNF-α insert was performed using 100 mM NaCl, 10 mM MgCl ₂ and 10 mM sodium PIPES (Sigma, USA) solution. 10 μg / ml (10 nM) DNA was heated to 95 ° C. and slowly cooled to room temperature prior to denaturation experiments. The temperature was raised at 0.5 ° C./3 minutes. Melting tests were performed on a diode array spectrophotometer (Hewlette Packard, USA) equipped with a Peltier temperature controller.

  RNA preparation

Total RNA preparation of normal and cancerous liver tissue was performed using Tri reagent (MRC, USA) according to the manufacturer's recommended protocol. The tissue was washed with phosphate buffered saline and sliced into small pieces. The sliced tissue was then homogenized in an optimal volume of Tri reagent for 10 minutes. Purification of poly (A) ⁺ mRNA was performed using a poly (A) quick mRNA isolation kit (Stratagene, USA) according to the manufacturer's instructions. Purified poly (A) ⁺ mRNA was used as a template for target DNA preparation.

  Target cDNA preparation and hybridization

The general procedure for hybridization was performed according to Dr. Patrick O. Brown's laboratory protocol (http://cmgm.stanford.edu/pbrown). Briefly, ² μg of each poly (A) ⁺ mRNA obtained from normal and tumor tissue of the liver was reverse transcribed using oligo-dT primers in the presence of Cy3-dUTP or Cy5-dUTP, respectively. The labeled cDNA was then purified with microcon-30. The purified target cDNA was resuspended in 80 μl of hybridization solution (3 × SSC and 0.3% SDS), denatured at 100 ° C. for 2 minutes, and applied to an array of LC sense molecules. Hybridization was performed in a humid chamber at 60 ° C. for 16 hours. Finally, each hybridized slide is washed once for 2 minutes with 2 × SSC, 5 minutes with 0.1 × SSC, 0.1% SDS, and 5 minutes with 0.1 × SSC, then at room temperature Spin dried prior to scanning.

  Data acquisition and analysis

  Fluorescent target cDNA hybridized to the cDNA microarray was detected by scanning the slide using a GenePix 4000B scanner (Axon instruments, USA). The PMT (photomultiplier tube) values for Cy3 or Cy5 were 450 and 500, respectively. The scanned image was then analyzed using the GenePix Pro3.0 software package. The signal intensity value was measured by subtracting the background median from the median intensity of each spot. Expression values were normalized with a single product sum normalization factor and applied to the total Cy5 / Cy3 ratio so that the central normalized Cy5 / Cy3 ratio was 1.0.

  Large-scale preparation of LC sense molecules

  The recombinant phagemid was transformed into competent E. coli infected with helper bacteriophage, M13K07. Transformed cells were incubated overnight at 37 ° C. on LB agar plates containing ampicillin (50 μg / ml). One colony was carefully isolated and seeded in LB liquid medium (10 g bactotryptone, 5 g yeast extract, 10 g / 1000 ml NaCl, 50 μg / ml ampicillin and 70 μg / ml kanamycin). The cells were then cultured for 14 hours at 37 ° C. with constant agitation. After centrifuging bacterial cells at 6,000 rpm for 10 minutes at room temperature, 100 ml of culture supernatant was mixed with Solution I (20% PEG8000 + 2.5M NaCl) and incubated at room temperature for 10 minutes. The sample was then placed in a column hole containing a borosilicate filter under vacuum for 10 minutes. For M13 lysis and binding, 50 ml of Solution II (4M NaClO4, 50 mM Tris-HCl, pH 8.5) was loaded onto the column and incubated for 10 minutes at room temperature for complete lysis of the bacteriophage. A vacuum was applied for 10 minutes to allow the filter to absorb the LC sense molecules. Then 100 ml of Solution III (80% EtOH, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5) was added to the column and evacuated for 10 minutes. The washing step with Solution III was repeated again and the buffer solution was removed by vacuum for an additional 15 minutes. LC sense molecules were eluted using 10 ml of sterile water. LC sense molecules were loaded on a 1% agarose gel and photographed under UV light for characterization and quantification.

  result

  Preparation of 1-LC sense array

  We prepared microarrays with LC-sense molecules (Figure 1) to test their utility in quantitative profiling of differential gene expression. Competent E. coli cells containing the helper bacteriophage M13K07 were transformed with 1,152 non-degenerate clones of recombinant pSPORT1 phagemids to produce LC sense molecules. High throughput production of LC sense molecules was achieved in a 96 well format. Purified LC sense molecules were electrophoresed on a 1% agarose gel and photographed with UV light (FIG. 2). A microarrayer was used to align single-stranded DNA samples of LC sense molecules on silanized glass slides.

  Melting point of 2-LC sense molecule

  Structural differences between single-stranded LC sense molecules and double-stranded phagemid DNA containing the TNF-α insert were tested by measuring the melting point (Tm1 / 2). When the absorbance at 260 nm was monitored with double-stranded phagemid DNA while gradually raising the temperature, a typical color change was detected at about 87 ° C. (FIG. 3A). However, when the single-chain LC sense molecule was tested for its melting point, a gentle gradient color change was detected at about 54 ° C., indicating the denaturation of the inner molecular short duplex (FIG. 3B). From these results, it was confirmed that the LC sense molecule was a single chain molecule. In addition, the optimal hybridization temperature was determined based on these results.

  3- Confirmation of RNA quality

RNA quality often determines the results of microarray experiments. Poly (A) ⁺ mRNA prepared from normal and cancerous liver tissue was used to synthesize Cy3-dUTP or Cy5-dUTP labeled cDNA, respectively. Labeled cDNA was mixed and hybridized at 65 ° C. to probe the cDNA seeded on the DNA chip. The LC sense microarray was washed, scanned with a scanning device (FIG. 4A), and analyzed with software. The data was then log2 transformed and then a scan plot (FIG. 4B). Scanned images demonstrated that the RNA quality was sufficiently pure to be used for labeling and hybridization to LC sense arrays.

  4-Identification of differentially expressed genes in cancerous liver tissue

Using poly (A) ⁺ mRNA prepared in Example 8.3 and confirming its strength, the labeled target cDNA is placed on an LC microarray and hybridization is performed at 60 ° C. Except, the same procedure as described above was used to detect the expression profile of genes in cancerous liver tissue. The LC sense microarray was then scanned and analyzed for expression profiles (FIG. 5). The data was then log2 transformed and then a scan plot (FIG. 6). Genes with a median of less than 200 were excluded from further data processing. From experiments, we found that 29 of 1,152 genes (up to 2.5%) were up-regulated in liver cancer tissue (Table 1). Among the 29 genes, in particular, the CD44 antigen (Endo K et al., J. Hepatology, 32 (1): 78-84, 2000), inosine monophosphate dehydrogenase (Jackson RC et al., Nature 256). (5515): 331-333, 1975), multiple endocrine hyperplasia 1 (Nakajima K et al., Intern. Med. 30 (1): 20-24, 2000) and calcium / calmodulin-dependent protein kinase 2 ( Arizono K et al., Life Sci, 53 (12): 1031-1037, 1993) has already been reported to be involved in liver cancer progression. On the other hand, 6 out of 1,152 genes were down-regulated in liver cancer tissues (Table 2). Among the 6 (~ 0.5%) genes, fibrinogen-like 1 (Kohno T et al., Jpn. J. Cancer Res. 91 (11): 1103-1110, 2000) is expressed in adults. It has already been reported that it is down-regulated in T cell leukemia. These results indicate that LC sense molecules can be used as binders in microarrays that detect genes that show differential expression.

  Large-scale preparation of 5-LC sense DNA

  Mass production of LC sense molecules will require the creation of a large number of DNA microarrays with consistently reliable quality. Mass production of LC-sense molecules has been achieved with a semi-automatic “prototype” device. The apparatus is equipped with a 96 purification column with an inner diameter of 37 mm, two 8-hole distributors with 30 or 100 ml pumping capacity, respectively, and a vacuum manifold (60 W × 42 L × 60 H). Recombinant phagemids were transformed into competent E. coli cells containing the helper bacteriophage M13K07. One colony was picked, inoculated into 100 ml of LB liquid medium and cultured for 14 hours at 37 ° C. with constant agitation. LC sense molecules were purified from 100 ml of culture supernatant using a semi-automatic purification apparatus. LC sense molecules prepared in large quantities were placed on a 1% agarose gel and tested for their properties and quantity (FIG. 7). About 200 μg of LC sense molecule was obtained from 100 ml of the culture supernatant using the above apparatus.

All references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.

List of upstream regulatory genes (> 2X) in cancerous liver tissue

List of downstream regulatory genes (> 2X) in cancerous liver tissue

1 shows a scheme for the production of single-stranded LC sense molecules. The target gene cDNA is cloned into the multiple cloning sites of the M13 phagemid vector. This construct, when infected with helper phage, M13KO7, allows for the rescue of single-stranded LC sense molecules of the target gene. : Shows mass production of LC sense molecules in small amounts. 1,152 transformants with recombinant pSPORT1 phagemid were incubated and infected with M13 helper bacteriophage in a 96 well format for high throughput mass production of LC sense molecules. After purification, LC sense molecules were run on a 1% agarose gel to test their quantity and nature. C: Control LC sense molecule without insert sequence. : Figures 3A-3B show the melting point profiles of double stranded plasmid molecules and LC sense molecules. Absorption was monitored every 0.5 ° C increase at 3 minute intervals while the temperature was increased from 30 ° C to 95 ° C. A: Tm1 / 2 profile of double-stranded phagemid containing TNF-α insert. B: Tm1 / 2 profile of the LC sense molecule containing the TNF-α sense insert sequence. : Figures 4A-4B show confirmation of RNA properties. The integrity of poly (A) ⁺ mRNA prepared from normal and cancerous liver tissue was tested. Cy3-dUTP or Cy5-dUTP labeled target cDNAs were mixed together and hybridized with PCR products on a cDNA chip. After hybridization, the cDNA chip was washed, scanned with a scanner, and analyzed with software. The data was then scatter plotted. A: Scanned image of cDNA chip, B: Scattered spot of expression profile. : Shows a scanned image of an LC sense array obtained from cancerous liver tissue. The PMT values for Cy3 and Cy5 were 450 and 500, respectively. Genes that are upstream regulated relative to normal tissue are shown in red, genes that are downstream regulated are shown in green, and yellow represents genes that show no change in expression. : Shows a scanning plot comparing expression profiles between normal and cancerous liver tissue. The expression profile is shown as a bivariate scan plot obtained from the LC sense array tested. Each spot was scan plotted according to its intensity after log2 transformation. : Shows an example of mass production of LC sense molecules. The transformant by recombinant phagemid was inoculated into 100 ml of 2 × LB liquid medium, and then cultured at 37 ° C. for 14 hours under constant stirring. LC sense molecules were obtained from 100 ml of culture supernatant containing recombinant bacteriophage using a specially designed semi-automatic purification apparatus. After preparation, LC sense molecules were photographed on a 1% agarose gel under UV light for quantification and quantification. Lane 1, LC sense molecule produced in large amount (40 ng), Lane 2, LC sense molecule produced in large amount (30 ng), and Lane 3, LC sense molecule produced in small amount (32 ng).

Claims (20)

Library containing individual LC sense molecules. The library according to claim 1, wherein the LC sense molecule comprises a vector sequence and a probe sequence, and the probe sequence is in the sense direction. The library according to claim 2, wherein the vector is a single-chain generating phagemid. 2. The library of claim 1, wherein the LC sense molecule is about 1,000 to about 20,000 nucleotides in length. The library of claim 4, wherein the individual LC sense molecules are separated from each other. The library according to claim 2, wherein the vector is pSPORT1, pBluescriptIISK (+/-) or KS (+/-), pGEM-f, M13mp, pCR2.1, pGL2 or pβgal. The library of claim 2, wherein the vector is an M13 bacteriophage, a f1 bacteriophage, or an fd bacteriophage. An array comprising a plurality of individual LC sense molecules stably bound to the surface of a support. 9. The array of claim 8, wherein the support comprises a coating layer of aminosilane, poly-L-lysine or aldehyde. 9. The array of claim 8, wherein the support is a glass slide, ceramic, inorganic-organic composite, flexible plastic film, silicone, metal or membrane. (I) inserting a nucleic acid fragment into a vector that produces a single-stranded vector;
(Ii) preparing a bacterial transformant by introducing a vector containing the insert into competent bacterial cells that make the bacterial transformant;
(Iii) infecting a transformant with a helper phage that produces an LC sense molecule;
(Iv) isolating the LC sense molecule from the culture supernatant of the transformant; and (v) aligning the LC sense molecule on the support surface;
9. The method of creating an array of claim 8, comprising: 12. The method of claim 11, wherein the nucleic acid fragment is inserted unidirectionally into the vector. (I) labeling DNA in the sample;
(Ii) contacting a sample containing labeled DNA with the array of claim 8;
(Iii) hybridize the labeled DNA in the sample with the LC sense molecules in the array; and (iv) measure the binding of the DNA to the LC sense molecules, where the presence of the signal on the array indicates the aligned LC Indicates the presence of DNA for the sense molecule,
Detecting the presence of DNA in the sample for a population of individual LC sense molecules in the array. 14. The method of claim 13, wherein the label is a streptavidin-alkaline phosphatase conjugate, chemiluminescent or chemiluminescent. 14. A method according to claim 13, wherein the label is Cy3 or Cy5. (I) labeling a first population of DNA obtained from a first sample;
(Ii) labeling the second population of DNA from the second sample with a different label;
(Iii) contacting a sample comprising a first population of labeled DNA with the array of claim 8;
(Iv) hybridizing a first population of labeled DNA in the sample with LC sense molecules in the array;
(V) contacting a sample comprising a second population of labeled DNA with the array of claim 8;
(Vi) hybridizing a second population of labeled DNA in the sample with LC sense molecules in the array; and (vii) measuring the binding of the labeled DNA to the LC sense molecules, where the presence of a signal on the array Indicates the presence of DNA,
A method for detecting the presence of DNA in two or more nucleic acid molecules. A gene expression analysis kit comprising the array of claim 8 and instructions for using the array to detect DNA in a sample. (I) a container containing a primer for producing a test nucleic acid;
(Ii) a container containing dNTP and / or rNTP;
(Iii) a container containing a post-DNA synthesis labeling reagent such as a chemically active derivative of a fluorescent dye;
(Iv) a container containing a DNA synthase;
(V) a container containing a buffer medium;
(Vi) a container containing a signal generating reagent and a detection reagent;
(Vii) The gene expression analysis kit according to claim 17, comprising instructions used for detecting DNA. Cytochrome P450, subfamily IIE (ethanol-inducible) (GenBank accession number J02843);
Transcription elongation factor A (SII) 1;
EST [H. sapiens] weakly similar to KIAA0206 (GenBank accession number AI193075);
1.3 kb mRNA of human skeletal muscle for tropomyosin (GenBank accession number AI7997037);
KIAA0701 protein (GenBank accession number AI7907037);
Transcription elongation factor S-II, hS-II-T1 mRNA (GenBank accession number NM_003195);
Deafness autosomal dominant 5 (GenBank accession no. AF073308);
KIAA1037 protein (GenBank accession number AI383628);
KIAA0375 gene product (GenBank accession number AB002373);
Prefordin 5 (GenBank accession number AA287397);
KIAA0710 gene product (GenBank accession number AB014610);
Paired homeodomain transcription factor 1 (GenBank accession number U70370);
Outer retina membrane protein 1 (GenBank accession number L07894);
EST (GenBank registration number Z39419);
MYC-related zinc finger protein (purine-binding transcription factor) (GenBank accession number M94046);
Ubiquinone conjugate enzyme E2L3 (GenBank accession number AJ000519);
New human genome mapping of chromosome 1 (GenBank accession number AL040438);
Homo sapiens clone 24421 mRNA sequence (GenBank accession number AF070641);
Homo sapiens mRNA; cDNA DKFZp566J2146 (GenBank accession number AL050081);
Chromosome condensation 1 analog (GenBank accession number NM_001268);
KIAA0902 protein (GenBank accession number AB020709);
Tyrosine kinase related protein 9 analog (A6 related protein) (GenBank accession number AI188660);
EST (S. serevisie) weakly similar to ORF YOR150w (GenBank accession number AI129433);
Transcription elongation factor B (SIII), polypeptide 2 (GenBank accession number AW327285) and cofactors required for Sp1 transcriptional activity, subunit 9 (GenBank accession number AA665998),
A method for measuring cancerous liver cells, comprising detecting upstream regulation compared to normal liver cells of a gene selected from the group consisting of: Transmembrane protease, serine 2 (GenBank accession number U75329);
PAC272L16, a human gene isolated from chromosome 1 (GenBank accession number AL023754), which is similar to a calcium / calmodulin-dependent protein kinase;
CASP2 and RIPK1 domains (GenBank accession number AA811130) containing adapters with death domains;
Ariadne homolog (GenBank accession number AL040708); and NADH dehydrogenase (ubiquinone) flavoprotein 1 (GenBank accession number AW250734),
A method of measuring cancerous liver cells comprising detecting downstream regulation of a gene selected from the group consisting of normal liver cells compared to normal liver cells.

Images