HUP0100697A2 - Solid-phase tips and uses relating thereto - Google Patents

Abstract

The subject of the invention is a solid-phase sample retention tip for synthesizing or detecting nucleic acids, a solid-phase sample retention structure, an arrangement of solid-phase sample retention structures, and a combination of a solid-phase sample retention structure and a microtiter plate. The subject of the invention are also methods for the production of a solid-phase sample retention tip for use in solid-phase molecular biological processes, and for converting a biomolecule into a solid-phase sample. HE

Description

PO 1 0 0 69 7

Representative:

Danube

Patent and Trademark Office Ltd.

Budapest

Tips for solid-phase studies and their applications

The present invention generally relates to solid-phase methods for the synthesis and analysis of nucleic acid molecules. More particularly, the present invention relates to improved solid-phase sample retention tips, supports, designs and arrangements for use in performing solid-phase methods that can be advantageously used in various biomolecular processes. The present invention therefore also relates to methods for preparing a solid-phase sample retention tip and for converting a biomolecule into a solid-phase sample.

Nucleic acid hybridization provides specific identification of biomolecules, such as DNA and RNA sequences, and has proven to be a powerful technique in medical diagnostics. For example, nucleic acid hybridization techniques have been used for the analysis of genetic polymorphisms, genetic diseases, cancer, and viral and microbial

Our file number: 92476-5456/SG for the detection of pathogens, screening of clones, and assembly of genomic fragments [for a review, see Chetverin and Kramer, Bio/Technology 12, 1093 (1994)]. The development of automated synthesis of oligonucleotide probes has also facilitated rapid, simple, and inexpensive diagnostic tests based on nucleic acid hybridization. The use of DNA probes in analytical procedures has been reviewed by Matthews and Kricka [Matthews and Kricka, Anal. Biochem. 169, 1 (1988), see also “DNA probes ed.: Keller and Mánk, 2nd ed., Stockton Press, (1993); Persing et al., “Diagnostic Molecular Microbiology, American Society for Microbiology (1993)].

The general implementation of nucleic acid hybridization involves first immobilizing the selected nucleic acid on a solid support, such as a nitrocellulose plate or nylon membrane, followed by hybridization with a nucleic acid suitable for detection. The disadvantage of such procedures is that the immobilized nucleic acid typically does not bind strongly, resulting in loss of the selected material from the support. This is also compounded by the fact that only a small amount of nucleic acid molecules are available for hybridization.

These problems can be overcome by the “sandwich-type” hybridization method. In the sandwich-type method, the selected nucleic acid is hybridized with a capture oligonucleotide covalently bound to a solid support. Then, the probe suitable for detection is hybridized with different parts of the captured nucleic acid, and the presence of the probe is measured.

The discovery of new therapeutic targets and diagnostic markers has been greatly facilitated by methods for analyzing gene expression patterns from large databases of expressed sequence regions [Fannon, Trends Biotechnoi., 14, 294 (1996)]. Such sequence data from a wide range of cDNAs provide a wealth of information for identifying genes of interest for drug development. Comparison of expression patterns between healthy and diseased tissues allows inferences to be made about gene function and the identification of therapeutically relevant genes that may be candidates for drug research and development programs.

A major obstacle to the widespread use of solid-phase cDNA synthesis and the use of DNA probes in simple assays is the lack of a solid support and a fixation method suitable for the hybridization process. The use of solid supports in DNA probe-based hybridization has been reviewed by Meinkoth and Wahl [Meinkoth and Wahl, Anal. Biochem. 138, 267 (1984)].

Poly(ethyleneimine) (“PEI”) coatings are widely used in biomolecule-binding methods. The binding capacity of PEI is very high for several reasons. For example, PEI is very hydrophilic, so it readily wets aqueous solutions containing biomolecules. In addition, PEI contains many amino groups that can form salts with acidic groups of biomolecules. However, the ability of PEI to absorb aqueous solutions of biomolecules is precisely why it has not been used to prepare biomolecule arrays. When aqueous solutions of biomolecules are placed on a PEI layer, the solution quickly permeates the PEI coating, rather than remaining in a specific, discrete location.

Spring probes have become widely known since their introduction at the beginning of the development of the printed circuit board manufacturing industry. These probes are mechanical devices that can meet the requirements of accuracy and reliability required for testing various electronic components and their connections when assembled into working circuit boards. Spring probes are essential electromechanical devices that typically consist of a plunger, ball, and piston enclosed in a tubular housing. Some probes are specifically designed to conduct current, while others are used to drill, crimp, and secure components to a circuit board, while others are used to perform soldering. We do not find any information regarding the design or sale of spring probes that would suggest that these devices, as mechanical devices suitable for transferring and sequencing solutions onto a solid support, would have potential applications in the fields of microbiology, biochemistry, or molecular biology.

Accordingly, there is a need for highly efficient, cost-effective means for sequencing biomolecules on solid supports. The present invention provides these and related advantages, as will be described in detail.

The solution according to the invention is summarized below.

The invention provides a solid-phase sample retention device that overcomes the technical obstacles previously encountered and provides additional related advantages.

In one embodiment of the invention, a solid-phase sample retention tip is provided for use in a method for synthesizing or identifying biomolecules. The sample retention tip comprises a solid support tip structure that is attachable to a support mandrel, and the tip structure, or at least a portion thereof, is coated with a chemical layer. In one embodiment of the invention, the tip structure is removably attached to the support mandrel. The tip is partially conical and has multiple grooves. These grooves engage multiple heat exchanger fins that allow the tip structure to be rapidly heated and cooled when thermocycling methods are used to synthesize or identify biomolecules.

In another embodiment of the invention, a sample retention structure is provided for solid-phase synthesis and/or identification of biomolecules. The sample retention structure includes a holding pin—the tip is connected to the holding pin—and a tip structure that is, or at least a portion of it, coated with a chemical layer. The chemical layer is adapted to bind the biomolecule, such that the biomolecule forms a solid-phase pattern on the tip structure. In one embodiment of the invention, the holding pin is a spring probe, or some other spring pin. The tip structure is made of a material belonging to the nylon 6/6 family and is removably connectable to the spring probe.

In another embodiment of the invention, a series of solid-phase sample retention assemblies are provided, wherein the support pins are connected to the base in a selected order. The end portion of each of the support pins is separated from the base and a plurality of tip structures are connected to the end portion. The chemical layer covers the tip structure, or at least a portion thereof. The chemical layer is capable of binding the biomolecule, such that the biomolecule forms a solid-phase sample on the tip structure.

According to another embodiment of the invention, a solid-phase sample retaining structure is combined with a microtiter plate. The well of the microtiter plate is designed to contain a defined volume of sample containing a biomolecule. The solid-phase sample retaining structure is sized so that the structure, or at least a portion thereof, extends into the well. The solid-phase sample retaining structure includes a support pin, the tip structure is connected to the support pin such that the tip structure can be movably positioned in the well of the microtiter plate, and the chemical layer covers the tip structure, or at least a portion thereof. The chemical layer is adapted to bind the biomolecule, such that the biomolecule forms a solid-phase pattern on the tip structure.

In one embodiment of the invention, the microtiter plate has a plurality of wells, and the solid phase sample retention structure includes a plurality of support pins arranged in a selected pattern, and the plurality of tip structures are connected to the support pins in a manner that provides a positionable arrangement of solid phase sample retention tips over the plurality of wells of the microtiter plate.

In another aspect of the invention, solid-phase sample retention tips are combined with a microtiter plate having a plurality of wells. The solid-phase sample retention tips are movably positioned within the wells of the microtiter plate. The microtiter plate and sample retention tips can be stored as a unit, so that the solid-phase sample on the sample retention tip can be easily stored as needed for the synthesis or analysis process.

In another aspect, the invention provides a method for manufacturing a solid-phase sample-retaining tip for use in solid-phase molecular biology methods. The method includes the steps of forming a base material into a tip configuration, which tip configuration is attachable to a support mandrel and which tip configuration, or at least a portion thereof, is covered with a chemical layer, which chemical layer binds a biomolecule such that the biomolecule forms a solid-phase sample on the tip configuration, and attaching the chemical layer to the base material. In one embodiment of the invention, the chemical layer is poly(ethyleneimine) and the base material is nylon 6/6, and attaching the chemical layer to the base material includes covalently attaching the poly(ethyleneimine) to the nylon 6/6.

Another embodiment of the invention is directed to converting a biomolecule into a solid-phase pattern. The method includes the steps of immersing a portion of a tip structure in a solution containing the biomolecule. The tip structure has a substrate-binding portion, which portion has a chemical coating to which the biomolecule can bind. The biomolecule is bound to the chemical layer, which biomolecule thus forms a solid-phase pattern on the tip structure, and then after the biomolecule has bound to the chemical layer, the tip structure is removed from the solution.

According to another aspect of the invention, the method includes the steps of storing the solid-phase tip assembly in the storage component, which storage occurs after the biomolecule has been bound to the chemical layer. According to one embodiment of the invention, the storage component is a microtiter plate having a hole therein. The storing step includes placing the tip assembly in the hole, which step occurs after the biomolecule has been bound to the chemical layer—and placing the microtiter plate and the tip assembly as a unit in the storage location.

Below is a brief description of the figures included in the description.

Figure 1 is an isometric view of a series of solid-phase sample retaining structures, in accordance with an embodiment of the invention.

Figure 2A is an enlarged cross-section of the solid-phase sample retaining structure, taken substantially along line 2-2 of Figure 1.

Figure 2B shows a cross-section of a solid-phase material retaining structure according to an alternative embodiment of the invention.

Figure 3 is a partially enlarged cross-sectional view of the tip of the solid-phase sample retaining structure shown in Figure 1.

Figure 4 is an enlarged cross-sectional view of the tip design, taken substantially along line 4-4 of Figure 3.

Figure 5 shows a side view of the array of Figure 1, drawn in solid lines, positioned above a microtiter plate containing a plurality of wells filled with biomolecule samples. The dashed lines show the state where the tip structures are positioned inside the wells in a lowered state.

Figure 6 shows a side view of the arrangement of Figure 1 in a state where the plurality of tip structures are positioned in the wells of the microtiter plate.

These and other aspects of the invention will become apparent from the following detailed description and the accompanying drawings. In addition, the various references mentioned above are incorporated herein by reference in their entirety.

The solution according to the invention is described in detail below.

In the following description, many technical terms are used in their broadest sense. The following definitions are provided to assist in understanding the invention.

The term "structural gene" refers to a nucleotide sequence that is transcribed into messenger RNA (mRNA), and the messenger RNA is translated into the amino acid sequence characteristic of a specific polypeptide.

As used herein, "nucleic acid" or "nucleic acid molecule" means any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotide, fragment produced by polymerase chain reaction (PCR), and any fragment produced by ligation, cleavage, endonuclease, or exonuclease. Nucleic acid monomers may be naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally occurring nucleotides (such as α-enantiomers of naturally occurring nucleotides), or combinations of both. In the case of modified nucleotides, the modification may be on the sugar moiety and/or the pyrimidine or purine base moiety. Modifications to the sugar moiety include, for example, the replacement of one or more hydroxyl groups with halogens, amines, alkyl, and azide groups, or the conversion of functional groups to ethers or esters. In addition, the entire sugar moiety may be replaced with spherical, or with similar structures in terms of charges, such as azasugars and carboxylated sugar analogs. Examples of modifications at the base include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other well-known heterocyclic substitutions. The nucleic acid monomers may be linked by phosphodiester linkages or analogous linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphorose. ¹ enoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanilide, phosphoramidate, and similar linkages. The term "nucleic acid" also includes so-called "peptide nucleic acids," which are formed by the association of naturally occurring or modified nucleic acid bases with a polyamide backbone. The nucleic acid may be single-stranded or double-stranded.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, a DNA molecule encoding interleukin-2 that has been isolated from the genomic DNA of a mammalian cell is considered an isolated DNA molecule. Another example of an isolated DNA molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism.

As used herein, the term "biomolecule" refers to a nucleic acid molecule or a polymer composed of amino acid(s) or amino acid analog(s).

As used herein, a "detectable tag" or "detectable label" refers to a molecule or atom that, when conjugated to a nucleic acid molecule, produces a label useful in detection methods. Such labels or labels include photoactive agents or dyes, radioisotopes, fluorescent agents, mass spectrometrically detectable labels, and other molecules and labeling fragments. Compounds suitable for fluorescent labeling include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Compounds suitable for chemiluminescent labeling include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalate esters. Bioluminescent compounds suitable for use as detectable labels include luciferin, luciferase and aequorin.

The term "complementary DNA (cDNA)" refers to a single-stranded DNA molecule that is produced from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to a portion of the mRNA is used to initiate reverse transcription. The term "cDNA" is also used by those skilled in the art to refer to a double-stranded DNA molecule that contains, in addition to such a single-stranded DNA molecule, its complementary DNA strand.

The term "expressed" refers to the biosynthesis of the product of a gene. For example, in the case of a structural gene, expression includes the transcription of the structural gene into mRNA and the translation of the mRNA into one or more polypeptides.

As used herein, a "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that is capable of autonomous replication in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease cleavage sites into which foreign nucleotide sequences or nucleotide sequences encoding marker genes useful for identifying and selecting cells transformed with the cloning vector can be inserted in a specific manner without losing the essential biological functions of the vector. The marker genes may typically be genes that confer tetracycline or ampicillin resistance.

As used herein, an "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Gene expression is typically controlled by a promoter and optionally at least one regulatory element. Such a regulatory element is said to be "operably linked" to the promoter.

Similarly, a regulatory element and a promoter are operably linked if the regulatory element influences the activity of the promoter.

The term "recombinant host" refers to any prokaryotic or eukaryotic cell that contains a cloning vector or expression vector. This term also includes prokaryotic or eukaryotic cells whose chromosome or genome contains cloned genes introduced by genetic engineering.

As used herein, the term "hybotrope" refers to an aqueous or organic solution of any chemical substance or mixture of chemical substances, containing buffers, chelators, salts and/or detergents, which changes the enthalpy of the nucleic acid duplex by at least 20% compared to that measured in a reference solution (0.165 M NaCl, 0.01 M Tris, pH 7.2, 5 mM EDTA and 0.1% SDS). That is, the energy content of the nucleic acid duplex is reduced. The reference, immobilized nucleotide is the sequence 5'GTCATACTCCTGCTTGCTGATCCACATCTG-3', as given in the sequence listing at ID No. 9, while the dissolved nucleotide is typically 5'TGTGGATCAGCAAGCAGGAGTATG-3' labeled at the 5'-end with a fluorochrome, such as Texas Red. sequence, which is the sequence given in the sequence listing at accession number 10, was used. The “helix to coil transition” (HCT) of the oligonucleotide duplex (24 nucleotides in length) is 25°C or less. The HCT is the difference between two temperatures at which 80% of the duplex is single-stranded and 20% of the duplex is single-stranded. For a solution to be defined as “hybotropic,” the average minimum slope is the first derivative of the HCT and is 2.4 1/°C expressed in units of [(80% single-stranded - 20% single-stranded)/25°C].

As used herein, "T _m is the temperature at which half of the nucleic acid duplex molecules are single-stranded. While T _m is determined for a solution, T _d of the duplex refers to the state when attached to a solid support. Both terms indicate the temperature at which half of the duplexes are single-stranded.

In the sense of the description, "stringency" is the acceptable percentage of mismatched base pairs during hybridization under given conditions.

As used herein, the term "discrimination" refers to the difference between the T _d of a duplex with perfect base pairing and a duplex containing mismatches.

As used herein, the term "discrimination temperature" refers to the temperature at which hybridization performed allows duplexes containing mismatches to be identified and distinguished from duplexes that are perfectly matched.

Solid carrier

The figures show an arrangement (10) according to an example of the solution according to the invention retaining 12 solid-phase samples.

- 16 - ; .

1, the assembly (10) includes a plurality of sample retaining structures (12) attached to a base (14). Each of the sample retaining structures (12) includes a holding pin (16) having one end (18) fixedly attached to the base (14) and the other end (22) of the holding pin (16) engaging a sample retaining tip (20). In an exemplary embodiment of the present invention, each sample retaining tip (20) is a solid support structure of Nylon 6/6. The Nylon 6/6 is coated with a layer of (24) poly(ethyleneimine) (PEI) or other selected chemical layer. The (24) PEI or other selected chemical layer is suitable for binding a selected biomolecule to form a solid phase sample. The solid phase sample is used in a method for synthesizing or detecting one or more nucleic acids.

The arrangement (10) of the present invention includes eight rows of twelve sample retaining structures (12) arranged substantially in parallel. The eight rows of sample retaining structures (12) define an arrangement of ninety-six (12) sample retaining structures, wherein the sample retaining structures (12) are equidistant from each other on a base (14). The sample retaining structures (12) are each of approximately equal length, such that the sample retaining tips (20) are equidistant from the base, thereby defining an arrangement of solid-phase sample retaining tip structures that are substantially coplanar. The sample retaining tips (20) are spaced apart to fit well into a conventional 96-well Cetus plate or a microtiter plate suitable for receiving and retaining liquid samples of biomolecules or nucleic acids. Although the exemplary arrangement of the present invention includes an 8*12 (12) sample retaining structure, alternative embodiments may have other configurations, including a 1*8 arrangement, a 1*12 arrangement, and a 4*12 arrangement, or possibly larger arrangements such as a 16*24 arrangement.

The ninety-six (20) sample retention tips, as an example of the solution according to the invention, are suitable for immersion in the wells of the Cetus plate containing biomolecules, such that the biomolecules chemically bond to the PEI layer (24). After the sample retention tips (20) are removed from the sample, the biomolecules adhere to the PEI layer (24), thereby forming a solid-phase sample. The sample retention tips (20) with the solid-phase sample thereon can then be used in synthesis or analytical processes, such as solid-phase nucleic acid assays, or - as described in much greater detail below - detection processes.

According to one embodiment of the invention, the arrangement (10) is operated by a robot or an automatic actuator. The basic configuration (14) is provided by the actuator.

Ls. =.:. J, ·” is attached to the fixture and the sample retaining structure (12) is placed over the base structure (14). During automated testing, the actuator quickly and accurately moves the assembly (10) to the selected, adjusted location or position according to the needs of the predetermined testing, synthesizing or analytical procedure. With the assembly (10) and the ninety-six solid-phase samples, such automated testing allows for significantly faster testing, synthesizing or analytical procedures.

In part, the arrangement (10) is eminently suitable for automated processes due to the support pin (16) of the sample retaining structure (12). As best seen in FIG. 2A, each of the support pins (16) is a spring-loaded probe, typically used for assembly and testing of electronic components, but these probes have been adapted for use in the present invention. The spring-loaded probe generally includes a housing (28) that includes a deflector member (30). The piston (32) is received in the housing (28) so that the first end of the piston (34) is located within the housing (28) opposite the deflector member (30), and the second end of the piston (36) is located outside the housing (28). In an example of the present invention, the deflector member (30) is a compression spring that biases the piston (32) toward the base (14). A flange (38) is provided at the first end of the piston (34) which abuts against a stop (40) to prevent the piston (32) from being radially ejected from the housing (28) and to limit the maximum travel of the piston (32) relative to the housing (28). The second end of the piston (36) is rigidly secured to the base (14) and the piston (32) extends outwardly in a direction substantially perpendicular to the base (14).

In an exemplary embodiment of the present invention, the housing (28) includes an inner cylinder (42) and an outer cylinder (44). The first end of the deflector (30) and the piston (34) are located within the inner cylinder (42). The outer cylinder (44) removably includes the inner cylinder (42) and frictionally engages the inner cylinder (42) so that the inner cylinder (42) and the outer cylinder (44) are removably secured to each other. The end of the outer cylinder (44) is the distal end (46) which is distal from the base (14) and which is connected to the tip (20). Accordingly, the outer cylinder (44) and the tip (20) can be removed as a unit from the inner cylinder (42) and the piston (32), which remain secured to the base (20). Thus, the outer cylinder (44) and the tip assembly (20) can be easily and quickly replaced as a unit without having to replace the entire spring probe. Suitable spring probes are available from Everett Charles (Pomona, California), Interconnect Devices (Kansas City, Kansas), Test Connections, Inc. (Upland, California), and other manufacturers. Although the example of the present invention uses a spring probe as the support pin (16), alternative embodiments of the invention may utilize other support pins, including opposed or non-opposed support pins.

As best seen in Figure 2B, in an alternative embodiment of the invention, the support pin (16) is a spring-loaded probe, but the probe is rotated 180° relative to the embodiment shown in Figure 2A and described above. For example, the distal end of the outer cylinder (44) (46) is rigidly attached to the base (14), and the second end of the piston (36) is located distal to the base (14) and is connected to the tip (20).

The spring probe provides protection against damage to the assembly (10) during operation. During sample collection or analytical procedures, for example, where the assembly (10) is positioned in a selected position and the tip (20) is inserted into the holes of the Cetus or similar plate, the support pin (16) or the tip (20) may inadvertently strike a surface or other object. In such a case, the spring probe is compressed in the axial direction, thereby cushioning the impact, and then returns to its original uncompressed position.

As best seen in Figure 3, in an exemplary embodiment of the present invention, the tip (20) is in the shape of a frustocone with a plurality of grooves or channels (50) formed therein. The tip (20) may be connected to the support mandrel (16) or may be integral with the support mandrel. The grooves (50) are "V-shaped grooves" extending axially between the uniform distal surface (48) and the uniform proximal surface (54). The ridges or ribs of the grooves (50) are held together at a defined angle from the proximal surface (54) to the distal surface (48). The frustoconical shape of the tip (20) was selected to fit virtually perfectly into the low-angle cross-sectional shape of the hole in the Cetus plate. Accordingly, the tip (20) was sized and shaped to fit into a very precisely defined position in the hole in the Cetus plate.

The tip assembly (20) includes an aperture (56) having an open proximal end (58) and a closed distal end (60) midway between the distal end (48) of the tip assembly (20) and the unitary proximal end (54). The aperture (56) is shaped and dimensioned to removably receive the distal end (46) of the support pin (16). Accordingly, the tip assembly (20) is removably connected to the support pin (16). However, the tip assembly may be permanently connected to the pin, and the pin and tip assembly may be a unitary structure.

In the illustrated embodiment of the invention, the mandrel housing (56) is coaxially coupled with the longitudinal axis of the tip assembly (20). The proximal portion (59) of the mandrel housing (56) is generally funnel-shaped, i.e., the mandrel housing (56) is open, the proximal end (58) having a larger diameter than the closed distal end (60). The proximal portion (59) is configured to receive the distal end (46) of the support mandrel (16). If, during assembly, the support mandrel (16) is slightly misaligned with respect to the mandrel housing (56), the funnel-shaped proximal portion (59) will receive and guide the support mandrel (16) into a position such that the spring probe is coaxially coupled with the longitudinal axis of the tip assembly (20).

As best shown in FIG. 4, in an exemplary embodiment of the invention, the mandrel housing (56) is defined by an inner wall about an axis (61) of generally circular cross-section. The distal end of the spring probe (46), however, is generally square in cross-section having four corners (63). The distal end of the spring probe (46) is configured so that the four corners (63) frictionally engage the inner wall about the axis (61) of the tip assembly (20) to frictionally retain the tip assembly (16) on the support mandrel (16).

In an alternative embodiment of the invention, the end portion has a polygonal cross-section with a plurality of corners that engage the inner wall of the tip assembly (61) about the axis. For example, the end portion has an octagonal cross-section with eight corners that frictionally engage the inner wall of the axis (61). In another alternative embodiment of the invention, the circular cross-section of the distal portion of the support pin is substantially the same as the circumference of the circular cross-section of the pin socket (56), such that the tip assembly (20) is press-fit to the distal end of the spring probe and is retained thereon by friction. In another alternative embodiment of the invention, the tip assembly (20) is bonded to the distal end of the spring probe (46) by a conventional adhesive, such that the tip assembly is permanently attached to the support pin (16).

As best shown in Figure 4, the grooves (50) and the ribs (52) define a truncated conical, generally star-shaped tip (20). As a result, the tip (20) has an enlarged outer surface (62) such that a greater amount of biomolecules can bind to the PEI layer (24) during solid-phase pattern formation. In an exemplary embodiment of the present invention, the distal surface (48) of the grooves (50) and the proximal surface (54) of the tip (20) define an enlarged solid support surface made of Nylon 6/6 material that is covalently bonded to the PEI layer (24). In an alternative embodiment of the invention, the tip structure (20) is made of a solid material, such as glass or silicone, and the PEI layer (24) is covalently bonded to this solid support by a silylation chemical process, as described in detail below.

In an alternative embodiment of the invention, the enlarged outer surface (62) of the tip (20) is recessed along the grooves (50) and ridges (52), thereby creating an additional increased surface area to which the PEI layer (24) is bonded. In one embodiment of the invention, the recesses are generally microscopic in size, and in an alternative embodiment of the invention, the recesses are macroscopic in size. Accordingly, with the recesses

- 24 (20) tip design, with an increased reaction surface area, allows for greater efficiency during synthetic or analytical procedures.

During certain selected synthetic and analytical processes, heat exchange occurs through the tip (20), in which the temperature of the tip (20) alternates between high and low values. The fins (52) of the tip (20) form a plurality of heat exchange fins (64) that allow the tip to change temperature more rapidly during thermocycling. As a result, thermocycling can be performed more quickly and more efficiently.

As best shown in Figure 5, the arrangement (10) is modified to be suitable for use with a microtiter plate (70) having a plurality of wells (72). As discussed above, a portion of the wells (72) are shaped to substantially fit the frustoconical shape of the tip assembly (20). Accordingly, the ribs (52) substantially fit the sidewalls (74) of the wells (72) and the tip assembly is flat, with the distal surface (48) positioned against the bottom of the wells (76). In a preferred embodiment of the invention, the wells of the microtiter plate (70) are arranged in substantially parallel rows of eight, twelve (72) wells, thus forming a ninety-six well configuration that can be mated with the arrangement of tip assemblies (10). According to another embodiment of the invention, the microtiter plate (70) may have a well arrangement

1*8 holes, 1*12 holes, and 4*12 holes, or perhaps a larger number than these, such as 16*24 holes.

In the use of the arrangement (10), the arrangement can be moved automatically or manually from a raised position, which is shown in solid lines in Figure 5, where the tip assembly (20) is lifted out of the wells (72), to a lowered position, which is shown in dashed lines in the figure, where the tip assembly is placed in the wells (72). In one example, the wells (72) contain a liquid sample containing the selected biomolecule. When the arrangement (10) is in the lowered position, i.e., the tip assembly (20) is in the liquid sample, a chemical reaction occurs between the PEI layer (24) and the biomolecule, resulting in the formation of a solid phase sample of the selected biomolecule. In the exemplary embodiment of the present invention, the depth of the well (72) is approximately 33% greater than the depth of the tip (20), so that when the tip is immersed in the well, the liquid sample will flood the entire tip, thereby binding as many biomolecules as possible.

As best shown in FIG. 6, the arrangement of the sample retaining structure (10) (12) is adapted to receive the tip assembly (20) in the well (72) as shown in solid lines, such that the tip assembly remains within the well. The base assembly (14) and the support pin (16) are then removed from the microtiter plate (70) as a unit. As a result, the ninety-six (20) tip assemblies in the wells (72) of the microtiter plate (70) remain as a unit or are stored, for example, in cold storage or other suitable storage areas, until the solid phase sample is required for the selected synthetic or analytical process.

In an exemplary embodiment of the present invention, with respect to the microtiter plate (70), the holes (72) hold the tip assemblies (20) in a very precisely defined position, such that the tip assemblies can be easily and substantially simultaneously mounted onto the support pins (16). For example, the microtiter plate (70) is held in a known and fixed position, and the base assembly (14) and support pins (16) are moved as a unit, either automatically or manually, into position over the holes (72) such that the support pins are substantially coaxially aligned in the tip assembly with the pin housing (56). The base assembly (14) and the support pins (16) are then moved as a unit toward the microtiter plate (70) to press the support pins (16) into the sockets of the tip assembly, thereby releasably engaging the tip assembly with the support pins. The base assembly (14), the support pins (16) and the tip assemblies (20) are then moved as a unit away from the microtiter plate (70), thereby removing the tip assemblies (20) from the wells (72). The sample retaining tip assemblies (12) containing the selected solid phase sample can be transported to a predetermined location and subjected to the selected solid phase nucleic acid analytical or synthetic process.

The solid supports of the invention can be used simultaneously and are preferably designed in a 96-well or 384-well format. In the case of a 96-well or 384-well design, the solid supports can be attached to legs, bases, or rods either detachably or, depending on the individual design, can be an integral part of the solid support. The individual design of the solid supports is not of particular importance for the operation of the assay, but rather has an impact on the automation of the assay process.

3. Methods for binding nucleic acid molecules to a solid support

The ideas described herein can be applied to a variety of methods that require the attachment of a nucleic acid molecule, peptide, polypeptide, or protein to a solid support. Examples of solid-phase assay and detection methods that can be implemented using these ideas are described below.

Standard methods can be used to attach nucleic acids to the tips. For example, nucleic acid molecules modified at the 5' end with aldehydes or carboxylic acids can be attached to a solid support containing hydrazide residues [see, e.g., Kremsky et al., Nucleic Acid Res. 15, 2891 (1987)]. Or, in a carbodiimide-coupled reaction, 5'aminohexyl phosphoramidate derivatives of oligonucleotides can be attached to a solid support containing carboxyl groups [see, e.g., Ghosh et al., Nucleic Acid Res. 15, 5353 (1987)].

The solid support is preferably coated with an amino polymer, such as poly(ethyleneimine), acrylamide, amine dendrimers, etc.

- are coated. Amines are used on polymers for the covalent immobilization of nucleic acids. As described herein, nucleic acids are preferably attached to the solid support by poly(ethyleneimine) coatings. The chemistry used to attach the PEI layer to the substrate depends substantially on the chemical properties of the substrate. In the state of the art, a number of suitable chemical processes are known for this purpose by which the PEI layer can be attached to the substrate. For example, a PEI layer can be attached to a Nylon 6/6 substrate by the process described by Van Ness et al. [Van Ness et al., Nucleic Acid Res., 19, 3345 (1991) ; and WO 94/00600. International Publication No. ]. A suitable process for applying the PEI layer to a solid support, glass or silicone, is described, for example, by Wasserman [Wasserman, Biotechnol. Bioeng. 22, 271 (1980)] and D'Souza [D'Souza, Biotechnoi. Lett. 8, 643 (1986)].

Preferably, the PEI layer is covalently bonded to the solid substrate. When the solid substrate is glass or silicone, the PEI layer can be covalently bonded to the substrate by a silylation process. For example, PEI containing reactive siloxy end groups is commercially available from Gelest, Inc. (Tullytown, PA). When such reactive PEI is brought into contact with a glass or silicone tip, after brief agitation, the PEI adheres to the substrate. Alternatively, a bifunctional silylating reagent can be used. According to this process, the glass or silicone substrate is treated with a bifunctional silylating reagent to provide the substrate with a reactive surface. The PEI is then brought into contact with the reactive surface and covalently bonded to the surface via the bifunctional reagent.

The PEI layer is advantageously used for immobilizing nucleic acids to nylon tips, as described herein. A suitable method for coating Nylon 6/6 with PEI has been described by Van Ness et al. (1991, Nucleic Acid Res., 19, 3345). In outline, the nylon substrate was ethylated with triethyloxonium tetrafluoroborate to generate amine-active imidate esters on the nylon surface. The activated nylon was then reacted with PEI to provide a polymer coating with an extensive amine surface. Oligonucleotides containing 5-aminohexyl tails were activated with 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride), and the modified oligonucleotides were covalently attached to the nylon surface via the triazine moiety.

Accordingly, preferred nucleic acid polymers are "amine-modified," i.e., modified to contain a primary amine at the 5-terminus of the nucleic acid polymer, preferably with one or more methylene groups between the primary amine portion of the nucleic acid polymer and the nucleic acid portion. The number of methylene groups is preferably 6.

Using standard techniques, nucleic acid molecules can be modified by the addition of amine moieties. For example, the products of a polymerase chain reaction can be provided with amine moieties using primers modified with 5'hexylamine. Nucleic acid duplexes can be prepared by adding aminoallyl-dUTP (Sigma, St.

Louis, MO) amines can be introduced into nucleic acids by various polymerases, such as terminal transferase and aminoallyl-dUTP, or by ligase catalysis for the ligation of short amine-containing nucleic acid polymers into nucleic acids.

Preferably, the nucleic acid polymer is activated before being brought into contact with the PEI coating. This can be easily accomplished by combining the nucleic acid polymer with an amine functional group and an amine reactive chemical, such as cyanuric chloride. For example, an excess of cyanuric chloride is added to a solution containing the nucleic acid polymer. Preferably, the arraying solution may contain a molar excess of cyanuric chloride of 10 to 1000 times the number of amines in the nucleic acid polymer. Thus, most of the amino-terminated nucleic acid polymers will react with one molecule of cyanuric chloride, so that the nucleic acid polymer will end in dichlorotriazine.

An advantageous feature of the invention is that reaction solutions containing biomolecules can be precipitated onto the PEI coating, even if the reaction solution contains a significant amount of multifunctional amine-reactive chemicals. This method provides a significant advantage over other methods, since in other methods the active agent used for coupling must be removed before the nucleic acid polymer and the PEI layer react.

If the nucleic acid polymer is double-stranded, both strands, or one of the two strands, contain a terminal amino group. For immobilization of the double-stranded nucleic acid polymer, the double-stranded nucleic acid polymer can be bound to the PEI coating via a terminal amino group. Since only one of the two strands is covalently bound to the PEI coating, the other strand can be removed under denaturing and washing conditions. This approach, in the present invention, provides a suitable method for obtaining a single-stranded nucleic acid polymer array. A double-stranded nucleic acid polymer can be obtained, for example, from a PCR reaction product.

Preferably, the array solution can be buffered with commonly used buffers such as sodium phosphate, sodium borate, sodium carbonate, or Tris-HCl. The preferred pH range of the array solution is from 7 to 9, with freshly prepared sodium borate ranging from pH 8.3 to pH 8.5.

The various methods discussed below require the oligonucleotides to be attached to a solid support according to the invention. Preferably, the oligonucleotides are synthesized with a 5'-amine (usually a hexylamine containing a six-carbon side chain and an amine at the end). Typically, the oligonucleotides are 15-50 nucleotides long and are activated with homo-bifunctional or hetero-bifunctional cross-linking reagents, such as cyanogen chloride. The activated oligonucleotides can be purified from excess cross-linking reagent (e.g. cyanogen chloride) by molecular filtration chromatography, if necessary. The activated oligonucleotides are then mixed with the solid support to form the covalent bond. Following covalent attachment of the oligonucleotides, the unreacted amines on the solid support are blocked (e.g. with succinyl anhydride) to remove the positive charge of the solid support.

Certain methods require biotinylated oligonucleotides that bind streptavidin, which streptavidin is in turn bound to a solid support. Methods for producing biotinylated nucleic acid molecules and for binding streptavidin to a support are well known to those skilled in the art. For example, Van Ness et al. [Van Ness et al., Nucleic Acid Res., 19, 3345 (1991)] describe a method for biotinylating oligonucleotides in which the oligonucleotides are treated with activated biotin. Optionally, biotinylated oligonucleotides can be prepared by using biotin-labeled dNTPs in the synthesis of oligonucleotides [see, for example, Short protocols in molecular biology, 3rd edition, pp. 12-23-25, ed. Ausubel et al., published by John Wiley & Sons, Inc. (1995)]. Biotinylation procedures for nucleic acids are well known in the art and are described, for example, in Avidin-Biotin chemistry: A handbook (Pierce Chemical Company, 1992). Standard procedures for attaching streptavidin to the tips of the present invention can be found, for example, in the following publications:

In rations: Das and Fox, Annu. Rev. Biophys. Bioeng. 8, 165 (1979); Wilcheck and Bayer, Anal. Biochem. 171, 1 (1983).

The methods described herein can also be used for the study of peptides. General guidelines for conjugating peptides, polypeptides, and proteins to solid supports include, for example, Wong, Chemistry of protein conjugation and crosslinking (CRC Press, Inc., 1991) and Partis et al. [Partis et al., J. Prot. Chem. 2, 263 (1983)]. The use of peptides and antibodies in solid phase methods is also known, for example, Vaughn et al., Nature Biotechn. 14, 309 (1996); and Huse et al., Science 246, 1275 (1989).

4. Application of solid support in cDNA synthesis

As discussed above, there is an increasing demand for the synthesis of cDNA on solid supports. These cDNA molecules can be used for the preparation of cDNA libraries, gene expression analysis and as probes in diagnostic procedures. The disclosed solid support design poses the following problems in the currently used cDNA technologies: the need for large amounts of RNA input, low sample throughput, many user operations, extraction and precipitation with organic materials, the vector affects the insert size and poor applicability. The solid-phase approach has the advantage that, compared to the solution-based cDNA synthesis with intermediate precipitation steps, it requires a large number of steps.

In this teaching, various approaches to the production of cDNA molecules on a specific solid support are described. The process is illustrated by the following general scheme. First, RNA is prepared by standard methods. In the experiment described in Example 1, total RNA is prepared by acid-guanidium phenol extraction using one of the well-known methods [see, for example, Short protocols in Molecular Biology, 3rd Edition, pp. 4-4-6, ed. Ausubel et al., published by John Wiley & Sons, Inc. (1995); Wu et al., Methods in gene biotechnology, pp. 33-34, CRC Press (1997)]. The messenger RNA (mRNA) is then captured on a solid support, e.g., oligonucleotides containing oligo(dT) tails.

Alternatively, in the case of cell lysis and hybridization, it is possible to use a simultaneous lysis-mRNA capture recipe using chaotropic reagents such as guanidine thiocyanate or guanidine hydrochloride. With this approach, only a small number of cells need to be lysed. According to the procedure, the DNA is removed by passing the cell solution through a glass fiber filter, the mRNA is captured on the solid support, and unbound materials are removed by washing. This avoids the material losses inherent in standard RNA preparation, which can be traced back to the series of steps of extraction with the organic phase and ethanol precipitation.

The carrier-bound RNA is used therapeutically to prepare the first strand of cDNA using standard techniques. The second strand is then synthesized, or an adapter is added to one end of the first strand of cDNA. For example, three dNGs can be added to the 3' end of the first strand, for example by terminal transferase. Alternatively, an adapter can be ligated to the 3' end of the cDNA, and the complementary primer is then hybridized to the adapter. The second strand is then synthesized using the first strand of cDNA as a template.

Alternatively, randomly selected or specific sequences of the bound RNA can be amplified using polymerase chain reaction (PCR). In short, PCR is a process that relies on a specialized polymerase. The specialized polymerase is able to synthesize a complementary strand to a given DNA strand in a mixture containing deoxyribonucleotides and two DNA primers, each about 20 bases long and overlapping the target sequence. The mixture is first heated to separate the double-stranded DNA strands containing the target sequence from each other, and then cooled so that the primers can bind to their complementary sequences on the separated strands. The polymerase then extends the primers into new, complementary strands. Repeated heating and cooling cycles multiply the target DNA exponentially, as each new double strand, after separation, becomes a new template for further synthesis. After about an hour, 20 PCR cycles can amplify the target sequence one million-fold. Standard protocols for performing PCR are well known to those skilled in the art [see, for example, Delidow et al., “Polymerase chain reaction: basic protocols, in: PCR protocols: current methods and applications, ΙΣΟ, ed. White, published by Humana Press, Inc. (1993); Short protocols in Molecular Biology, 3rd Edition, 15-1 to 15-40, ed. Ausubel et al., published by John Wiley & Sons, Inc. (1995)].

Accordingly, a primer complementary to a known portion of the bound RNA can be added. It can then be amplified using a specific primer and a primer complementary to the adaptor. After amplification, the resulting DNA fragments can be cloned and the 5' end of the sequence can be determined. It is then possible to synthesize a new, hybrid primer that contains a sequence complementary to the adaptor and the 5' end of the gene. The full-length cDNA can then be amplified from the solid support.

A modification of PCR, called anchor PCR, allows the amplification of full-length mRNA, even when only a small amount of sequence information is available [Short protocols in Molecular Biology, 3rd Edition, pp. 15-27 to 15-32, ed. Ausubel et al., ed.:

- 37 John Wiley & Sons, Inc. (1995)]. This procedure requires an oligo(dT) primer that is complementary to the poly(A) tail of the mature mRNA when amplified to a known sequence in the 3' direction, or to the synthetic homopolymer tail added to the cDNA after synthesis of the first strand when amplified to a known sequence in the 5' direction.

These general cDNA synthesis procedures have at least two branch points, which provide an opportunity for further techniques that take advantage of solid support methodologies. The first branch point occurs after the synthesis of the first strand of cDNA. At this point, the remaining RNA template can be digested with RnaseH, hydrolyzed with sodium hydroxide, or removed by heat denaturation, and the single-stranded DNA template, guided by the oligonucleotide, can then be used to synthesize the second strand, for PCR, to prepare random primers, or for gene expression studies with labeled nucleotides. The ligated cDNA can also be used to prepare a subtraction library or probes for various applications.

A second branch point in solid-support cDNA technology may be the synthesis of the second strand of cDNA. Here, the choice is made between ligating an adapter to the cDNA, which enables processes such as the production of full-length, single-stranded cDNA probes, library production, in vitro transcription, and 5'-RACE.

An important advantage of solid-support-based cDNA synthesis is its ability to be automated. For high-throughput cDNA library preparation or gene expression studies, it is important to convert the solid support described here into a 96-well format. A robotic arm can be used to transfer 96 supports onto 96 gold-coated pins and direct cDNA synthesis in a standard 96-well Cetus plate.

5. Gene expression analysis

The solid support described herein can be used in high-throughput methods for assaying the expression of a large number of genes (1-2000) in a single assay. Such methods can be performed simultaneously with more than one hundred samples per run. The method can be used in drug screening, developmental biology, molecular studies of therapeutic agents, and the like. Thus, in one aspect of the invention, the invention provides a method for assaying the pattern of gene expression from a selected biological sample. The method includes the following steps: (a) detecting nucleic acids from a biological sample, (b) combining the detected nucleic acids with one or more selected detectably labeled nucleic acid probes under conditions and for a time suitable for the probes to hybridize to the nucleic acid, and wherein the identifiable label can be correlated with a particular nucleic acid probe and detected by spectrometry or potentiometry, (c) separating the hybridized probes from the unhybridized probes, (d) detecting the label by spectrometry or potentiometry, and (e) determining the gene expression pattern of the biological sample therefrom.

In a particularly preferred embodiment of the invention, assays or methods are provided that can be performed as follows. RNA from a target source is bound to a solid support via a specific hybridization step (e.g., capture of poly(A) mRNA with a tethered oligo(dT) capture probe). The solid support is then washed and cENS is synthesized on the solid support using standard techniques (e.g., reverse transcriptase). The RNA strand is removed by hydrolysis. The result is a population of DNA covalently immobilized on the solid support that reflects the diversity, abundance, and complexity of the RNA from which the cDNA was synthesized. The solid support is then hybridized with one to a few thousand probes that are complementary to the sequence of the gene of interest. Each probe is labeled with a detectable label by spectrometric methods, such as mass spectrometry. Following the assay step, excess or unhybridized probes are removed by washing, and a solid support, such as a microtiter plate, is placed in the wells and the detectable label is cleaved from the support. The solid support is then removed from the sample well and the contents of the well are measured by spectrometry. The appearance of the specific label indicates the presence of RNA in the sample and serves as evidence that the specific gene is expressed in the given biological sample. The method is also suitable for quantitative detection.

Compositions and methods for rapid measurement of gene expression are discussed in detail below. In general, the RNA source may be tissues, primary or transformed cell lines, isolated or purified cell types, or any other biological material in which determination of gene expression is useful. In the preferred method, the biological material is lysed in the presence of a chaotropic agent used to inhibit nucleases and proteases and to tightly hybridize the target nucleic acid to the support. Tissues, cells, and biological sources can be effectively lysed with a 1-6 molar chaotropic salt (guanidine hydrochloride, guanidine thiocyanate, sodium perchlorate, etc.).

After the biological sample is lysed, the solution is stirred for 15 minutes to several hours to effectively immobilize the target nucleic acid. Typically, capture of the target nucleic acid is achieved by base pairing the target RNA complement and immobilizing the capture probe on a solid support. One variant utilizes the 3 ^f -poly(A) extension, found in most eukaryotic mRNAs, to hybridize with oligo(dT) bound to the solid support. Another variant utilizes a specific or long (>50 bases) probe to capture RNA containing a specific sequence.

Another option is to use degenerate primers (oligonucleotides), which are primers that are effective in capturing a number of related sequences in the target RNA population. For example, RNA samples can be reverse transcribed with each of the following four degenerate, anchored oligo(dT) primer sets, the sequence of which is 5' T ¹² MN-3', where M can be G, A, or C and N can be G, A, T, and C [Short protocols in Molecular Biology, 3rd Edition, pp. 15-35 to 15-40, eds. Ausubel et al., published by John Wiley & Sons, Inc. (1995)]. Each primer set is defined by a degenerate 3' base at the M site.

The time required for hybridization depends on the complexity of the RNA sequence and the type of probe used. The hybridization temperature is determined by the chaotrope used and the final concentration of the chaotrope. General guidelines are available, for example, from the publication by Van Ness and Chen [Van Ness and Chen, Nucleic Acid Res. 19, 5143 (1991)]. In order to influence the diffusion of the target RNA, the lysate is preferably continuously mixed with the solid support. After the target nucleic acid has been captured, the lysate is washed from the solid support and all chaotropes or hybridization solutions are removed. The solid support is preferably washed with solutions containing ionic or non-ionic detergents, buffers and salts.

The next step is to synthesize DNA complementary to the captured RNA, in which the bound oligonucleotide serves as an extended primer (extension primer) for the reverse transcriptase. The reaction is usually carried out at 25-37°C and is preferably stirred during the polymerization reaction. After the cDNA has been synthesized, it is covalently bound to the solid support, since the extended primer is already an oligonucleotide bound to the solid support. The RNA is then hydrolyzed from the cDNA/RNA duplex. This step can be influenced by heating - heating denatures the duplex - or by using a base (e.g. 0.1 N NaOH) - the base chemically hydrolyzes the RNA. The purpose of this step is to make the cDNA available for subsequent hybridization with specific probes. The solid support, or series of solid supports, is then further washed to remove the RNA or RNA fragments. At this point, the solid support contains a population of DNA molecules approximately representative of the RNA population, which population corresponds to the RNA population in terms of sequence abundance, complexity, and distribution.

The next step is to hybridize the selected probes to the solid support in order to identify the presence or absence and relative abundance of a specific cDNA sequence. The probes are preferably oligonucleotides of 15-50 nucleotides in length. The sequence of the probes is determined by the end user of the assay. For example, if the end user wishes to study gene expression in an inflammatory process in a tissue, it is advisable to select probes that are complementary to several cytokine mRNAs, RNAs encoding lipid-modifying enzymes, or RNAs encoding factors that regulate cells involved in the inflammatory response, etc. Once the sequence set required for the experiments has been determined, an oligonucleotide probe is designed for each of the sequences, and a specifically cleaved region is designated for each probe. This region is then annealed to the appropriate oligonucleotide(s). The oligonucleotides are then hybridized to the cDNA on the solid support under appropriate hybridization conditions. After the hybridization step is complete, the solid support is washed to remove any unhybridized probe. The solid support or series of supports is then placed in a solution that affects the cleavage of the detectable region. The presence or absence of an expressed RNA is determined by measuring the amount of the detectable region. For example, regions detectable by mass spectrometry are analyzed with a mass spectrometer.

There are many variations of the differential expression analysis procedure described above. For example, differential expression can be examined using a subtracted library. One of the desired capabilities of any gene discovery program is to eliminate redundant responses in detecting gene expression patterns characteristic of a particular stage of activation or development. There are many recipes for preparing subtracted cDNA libraries, but most of them require large amounts of RNA or a pre-existing cDNA library. The use of a solid phase to capture inRNA allows the omission of a source of information corresponding to background. The desired mRNA species are reverse transcribed, and the RNA templates are degraded with alkali. The resulting “subtraction template” can be reused indefinitely.

To prepare a subtraction library, RNA from the source under investigation is heat denatured and hybridized to the first strand of cDNA on the subtraction template. Unbound RNA is then washed away, and subtracted RNA, either directly captured or hybridized after binding of all RNA, is eluted from the subtraction template. Following capture, cDNA is synthesized as previously described.

In a related approach, subtraction cDNA probes are prepared by hybridizing single-stranded cDNA from one cell type to immobilized mRNA from a closely related cell type and isolating a small portion of the unhybridized cDNA. As a result of this enrichment, DNA fragments of the subtraction cDNA can be used to identify cDNA clones containing differentially expressed sequences. The subtraction cDNA can also be used to construct subtraction cDNA libraries. Subtractive cDNA for testing or cloning purposes can be amplified by PCR [see, for example, Kuel and Battey, “Generation of a polymerase chain reaction renewable source of subtractive cDNA” in: “PCR protocols: current methods and applications,” ed.: White, ed.: Humana Press, Inc. (1993); Wu et al., “Methods in gene biotechnology,” 29–65, CRC Press (1997)].

In a further embodiment, a degenerate primer, biotinylated oligo(dT) as described above, is used to bind mRNA to a solid support and initiate first-strand synthesis [see, for example, Rosok et al., BioTechniques 21, 114 (1996)]. Following reverse transcription, the solid-phase cDNA is used as a template in a polymerase chain reaction with oligo(dT)MN and an optional decaliter as a primer. PCR products from two cell populations are compared after separation by polyacrylamide gel electrophoresis, the PCR bands of interest are eluted from the gel, and the purified PCR products are used as templates in a further polymerase chain reaction to amplify the selected bands. The products of the second polymerase chain reaction are then used as probes or further examined by sequence analysis.

6, Solid-phase diagnostic assay (A) Detection of polymorphism

Restriction endonucleases recognize short DNA sequences and cleave the DNA at specific sites. Some restriction enzymes cleave DNA at very rare sites, producing a small number of large fragments (ranging from a few thousand to a few million base pairs). Most restriction enzymes cleave DNA much more frequently, producing a large number of small fragments (ranging from less than a hundred to more than a thousand base pairs). On average, digestions performed with restriction enzymes with four base recognition sites produce fragments 256 bases long, those with six base recognition sites produce fragments 4000 bases long, and those with eight base recognition sites produce fragments 64000 bases long. Since hundreds of different restriction enzymes are now known, DNA can be cleaved into several different small fragments.

Although some known human DNA polymorphisms are based on insertions, deletions, or other rearrangements of non-repetitive sequences, the majority are based on either single base substitutions or changes in the number of tandem repeats. Base substitutions occur very frequently, on average once every 200–500 base pairs, in the human genome. Variation in the length of tandem repeat blocks is also common in genomes with at least tens of thousands of polymorphic sites, or “loci.” The length of the repeat blocks of a tandem repeat polymorphism ranges from one base pair in the (dA) _n (dT) _n sequences to at least 170 base pairs in α-satellite DNA. Tandem repeat polymorphisms can be divided into two groups. In the minisatellite/variable number tandem repeats (VNTRs), the total length of the repeat units is a few tens of thousands of base pairs and the typical repeat length is a few tens of base pairs, while in the microsatellite group, the length of the repeat blocks is a maximum of 70 base pairs and the repeat length is up to six base pairs. Most microsatellite polymorphisms identified to date are in the (dC-dA) _n or (dG-dT) _n dinucleotide repeats. sequence. Microsatellite polymorphism analysis involves the amplification of a small DNA fragment containing a block of repeats by polymerase chain reaction and subsequent electrophoresis of the amplified DNA on a denaturing polyacrylamide gel. The PCR primers are complementary to the sequences flanking the block of repeats. Traditionally, polyacrylamide gels are used rather than agarose gels because alleles often differ by only a single repeat size.

A variety of methods have been developed to examine DNA polymorphism. The most widely used method, the restriction fragment length polymorphism (RFLP) approach, is a combination of restriction enzyme digestion, gel electrophoresis, membrane blotting, and hybridization with a cloned DNA probe. The RFLP approach can be used to analyze base substitutions when the sequence change falls within a restriction enzyme recognition site, or to analyze minisatellites/VNTRs by selecting an enzyme whose cleavage site cleaves outside the repeat unit. Agarose gels generally do not provide sufficient resolution to distinguish between minisatellite/VNTR alleles that differ by only one repeat unit, but minisatellites/VNTRs are so variable that easily identifiable markers can still be found [Vos et al., Nucleic Acid Res. 23_, 4407 (1995)].

Solid-phase techniques have increased the ability to detect polymorphism. For example, using a biotinylated primer in conjunction with an allele-specific PCR primer, one form of an allele can be amplified. After amplification, the PCR products can be detected by hybridization in solution to a fluorophore probe, followed by capture of the hybrids with streptavidin immobilized on a solid-phase support [see, for example, Syvanen and Landegren, Human Mutation 3, 172 (1994)].

In another approach, called "solid-phase minisequencing," a biotinylated amplification product is immobilized on a streptavidin-coated support. Then, using the amplification product as a template, a sequence-specific extension reaction is performed with a separate nucleotide triphosphate complementary to one of the sequence variants to be isolated [see, for example, Syvanen and Landegren, Human Mutation 3, 172 (1994); Syvanen, Clin. Chern. Acta 226, 225 (1994); Járvelá et al., J. Med. Genet. 33, 1041 (1996)].

In an "oligonucleotide ligation assay," two differently labeled allele-specific oligonucleotides are compared for their ability to ligate to a 3' oligonucleotide [see, e.g., Nickerson et al., Proc. Natl. Acad. Sci. USA 87, 8923 (1990); Nickerson et al., Genomics 12, 377 (1992)]. The unlabeled oligonucleotide is immobilized on an avidin-coated solid support placed in a test tube. The presence of the target sequence is determined by measuring the signal generated by the bound labeled oligonucleotide. For example, the allele-specific oligonucleotide can be labeled with different fluorophores and the presence of the target is determined by measuring fluorescence.

Nepom et al. [Nepom et al., J. Rheumatol. 23, 5 (1996)] describe a method for genotype analysis in which the selected sequence is amplified by PCR in the presence of biologically derived nucleic acid and a biotinylated primer. A small portion of the amplified product is transferred to an automated device that performs allele-specific hybridization and detection.

Another possible way to detect polymorphism is by DNA fingerprinting. There are several methods developed for DNA fingerprinting, most of which use PCR to generate fragments [see, for example, Jeffries et al., Nature 314, 67 (1985); Wels and McClelland, Nucleic Acid Res. 19, 861 (1991)]. The choice of technique for DNA fingerprinting depends on the application (e.g., DNA typing, DNA marker mapping) and the organism under study (e.g., prokaryotes, plants, animals, humans).

In general, DNA imprinting can be accomplished by synthesizing full-length eDNA on a tip of the invention. The cDNA is then digested with a restriction endonuclease and unbound material is washed off the tip. Adapters are ligated to the bound cDNA, and the product is amplified and analyzed.

In the past few years, several fingerprinting techniques have been developed, including random amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), and arbitrarily primed PCR (AP-PCR). All of these techniques are based on the amplification of randomly selected DNA fragments of genomic origin with arbitrarily selected PCR primers. DNA fragments can be generated from any DNA without prior knowledge of the sequence. The pattern produced depends on the sequence of the PCR primers and the nature of the DNA used as a template. PCR is performed at low annealing temperatures to allow primers to anneal to multiple loci on the DNA. DNA fragments are generated when the primer binding sites are within a distance that allows amplification. In principle, a single primer is sufficient to produce banding patterns.

A much newer technique for DNA fingerprinting to detect polymorphism is the amplified fragment length polymorphism (AFLP) technique [Vos et al., Nucleic Acid Res. 23, 4407 (1995); Schr iner et al., J. Immunol.

Methods 196, 93 (1996)]. In outline, genomic DNA is digested with restriction endonucleases, ligated with oligonucleotide adapters. PCR ensures the selective amplification of restriction fragment sequences, and then, after fractionation by polyacrylamide gel electrophoresis, the amplified fragments are analyzed.

This method can be easily adapted to solid phase by using the tip of the invention. Here, full-length genomic DNA is bivalently immobilized on the tip. The bound DNA is then digested with a restriction endonuclease and the unbound material is washed off the tip. Adapters are then ligated to the immobilized DNA fragment and the bound DNA molecule is amplified and analyzed.

The AFLP technique has also been used for mRNA fingerprinting [Habu et al., Biochim. Biophis. Res. Commun. 234, 516 (1997)]. In this approach, double-stranded DNA was synthesized with anchored oligo(dT) primers and digested with TagI, which recognizes a four-base sequence. Then, TagI adapter was ligated to the ends of the cDNA fragments, and the fragments were amplified by PCR with selected primers according to general procedures for AFLP-based genomic fingerprinting. Preferably, mRNA fingerprinting is performed using the AFLP technique with oligo(dT) primers bound to a solid support as described herein.

Mutations can also be identified by their destabilizing effect on hybridization of the mutants to short oligonucleotide probes prepared for the target sequence [see, for example, Habu et al., Biochim. Biophis. Res. Commun. 234, 516 (1997)]. In general, this technique of allele-specific oligonucleotide hybridization involves amplification of the target sequence and subsequent hybridization with short oligonucleotide probes. An amplified product can be screened for many possible sequence variants by examining the hybridization pattern of the product with a series of immobilized oligonucleotide probes. An illustration of this approach is provided in Examples 6 and 7.

(B) General diagnostic procedures

DNA probes are useful for detecting the presence of infectious agents or diseased cells, such as tumor cells that express tumor-associated antigens. Typically, the biological sample to be tested is lysed with ionic detergents or chaotropic agents to release the target nucleic acids. Typical target nucleic acids include mRNA, genomic DNA, plasmid DNA or RNA, and rRNA (ribosomal RNA), viral DNA or RNA. To effect detection of the target nucleic acid, the target nucleic acid must be immobilized in some way. For example, the nucleic acids are immobilized on a solid support or substrate that exhibits some affinity for the nucleic acid. The solid support is then probed with labeled oligonucleotides prepared based on a predetermined sequence to identify the target nucleic acid of interest. The unhybridized probe is removed during a washing step, and the label is cleaved from the corresponding probe and measured (for a review, see Reischl and Kochanowski, Mol. Biotech. 3_, 55 (1995)).

In a general type of assay, oligonucleotides representing a characteristic region of the amplified sequence are bound to a solid support. The bond may be covalent, biotin-streptavidin, or similar. The target nucleic acid is used as a template to generate detectably labeled PCR products. These PCR products are hybridized to the capture oligonucleotides, and the presence of the PCR product is determined by a detection reaction based on the present invention.

In a variation of this approach, biotin-labeled PCR products are immobilized on a streptavidin-coated solid support. The immobilized PCR products are hybridized with a probe complementary to the labeled internal sequences of the amplified product.

As a further illustration of a diagnostic method, Wilber [Wilber, Immunol. Invest. 2 6, 9 (1997)] describes a solid-phase nucleic acid hybridization assay based on branched-DNA signal amplification techniques. In this assay, the presence of HIV RNA in plasma was detected by hybridizing multiple oligonucleotides to the target. Ten of the oligonucleotides bound to the surface of the target, while 39 oligonucleotides mediated hybridization between the branched DNA molecule and the RNA pol region. The probes with detectable labels bound to each arm of the branched DNA molecule.

Additional detection methods are well known to those skilled in the art. For example, a method suitable for solid phase detection may be enhanced colorimetry, such as an alkaline phosphatase system, a streptavidin system, or a horseradish peroxidase system. Radiometric detection is another alternative. Radioactive labels suitable for radiometric detection include the following isotopes: ¹ H, ¹²⁵ I, ¹³¹ I, ³⁵ S, ¹⁴ C, ³² P, and the like. Fluorescently labeled molecules provide additional detection options.

The invention, having been thus broadly described, will be better understood by reference to the following examples. The examples are for illustrative purposes only and are not intended to limit the invention.

Examples (A) RNA capture

For these experiments, total RNA was prepared by acid-guanidinium-phenol extraction using standard techniques [see, for example, Short protocols in Molecular Biology, 3rd Edition, pp. 4-4-6, ed. Ausubel et al., published by John Wiley & Sons, Inc. (1995)]. Polyadenylated RNA was attached to the tips by first heating the mixture containing the isolated RNA to 70°C, adding high-salt hybridization solution to the RNA, then adding the RNA to the tip, i.e., the solid support containing oligo(dT), and then placing the solid support in a shaker, such as a rotary mixer. The necessary mixing was achieved using various devices, including continuous vortexing, horizontal shakers, rotary shakers, and hybridization oven equipped with a shaking device.

We found that the time required for RNA capture depends on the amount of RNA introduced. At room temperature, 10 pg of total RNA is sufficient to reach 90% saturation of the tip in approximately two hours. In a typical resting cell, this amount of total RNA corresponds to approximately 100 ng of bound poly(A)+mRNA per tip. Using 30 pg of total RNA from the same source, similar saturation can be achieved in 30 minutes.

In another set of experiments using a human T-cell line as the RNA source, similar saturation was achieved within 1 hour after 10 pg of RNA under similar capture conditions. Several other RNAs were captured, including mouse, hamster, and human cell line RNAs, and all of these fell within the range of 90% saturation at 10 pg RNA/1-2 hours, thus defining the maximum and minimum incubation times.

(B) Synthesis of the first cDNA strand

After capturing poly(A)+ mRNA, the tip was washed three times in hybridization buffer to remove unbound RNA. Reverse transcription was performed in a volume of 30 µl,

It was performed in an optimized buffer system containing MMLV reverse transcriptase for 1-2 hours at 42°C on a rotating rack of a hybridization oven. As with the capture step, continuous mixing of the reagents is required for efficient first-strand synthesis.

In the initial experiments, 50100 ng of cDNA were produced per solid support. The product obtained during reverse transcription, i.e. the labeled double-stranded cDNA, was cleaved from the tip and separated by size by agarose gel electrophoresis and then indirectly examined by autoradiography. In several experiments, the length of the resulting RNA copies was similar to, or often longer than, those produced by conventional methods. The size distribution of the cDNA ranged from 0.5 to 20 kilobases, with a median of approximately 2.0 kilobases.

(C) Leveling of the second cDNA strand

After reverse transcription, the tip was washed three times to remove reagents and enzyme. Second strand synthesis was performed in a volume of 40 μΐ with the addition of one unit of RnaseH / 25 units of E. coli polymerase I. The reaction mixture was incubated at room temperature on a rotary shaker for 6-12 hours.

The mismatched extended ends were blunted by removing the second strand reaction mixture and adding T4 DNA polymerase and dNTPs before the ligation step. Incubation was performed for 30 minutes at 37°C in a hybridization oven on a rotator. To identify the products, the reaction was performed in the presence of ³² P-labeled dNTPs. The products formed during the synthesis of the second strand were either boiled or cleaved from the gel support with the restriction enzyme Ascl. The radiolabeled products were run on a gel and film was placed on it. Depending on the RNA selected, it is usually possible to obtain 50-120 ng of double-stranded cDNA with 10 pg of total RNA.

Following reverse transcription with an enzyme exhibiting RNaseH activity, such as MMLVRT, a thermostable DNA polymerase can be used. In this case, the thermostable polymerase digests the RNA template, eliminating the need for RNA removal in the final denaturation step.

An experiment comparing RnaseH/DNA pol I and Tthl DNA pol interase during second strand synthesis showed that there was little difference in the quantity of AscI-cleaved products and the quality of the products (checked by PCR amplification of selected genes such as GAPDH and IL-2). By using Tthl, the incubation time can be reduced from the 6 hours required at room temperature to 1 hour at 70°C. In the presence of manganese ions, Tthl polymerase also exhibits reverse transcriptase activity, but due to the high temperature and the presence of divalent cations, the incubation time must be very short to avoid possible RNA hydrolysis. Another advantage of performing DNA polymerization at high temperatures is that secondary structures are weakened, which facilitates the synthesis of very complex information.

(D) Adapter ligation - ligation to the vector

The semi-phosphorylated adapters were ligated to the double-stranded cDNA in a volume of 30 μΐ at a molar ratio of adapter/cDNA ends of 5-10:1. The preferred ligation buffer contained 10% PEG. The adapter-cDNA mixture was incubated with T4 DNA ligase at room temperature for half a day on a rotary shaker. The solid support was then washed three times to remove excess linkers. Following ligation, the 5'-hydroxyl groups on the adapters were phosphorylated with T4 polynucleotide kinase in the presence of ATP for one hour at 37°C on a rotating rack in a hybridization oven. The reaction was stopped by washing the tip three times with TE buffer. If the cDNA is to be used for other purposes, the phosphorylation step is not necessarily necessary.

In some applications, second-strand synthesis has been specifically initiated with an adapter immediately after reverse transcription. In this approach, after mRNA hydrolysis, a partially single-stranded heteroduplex adapter can be ligated with T4 RNA ligase. The partially double-stranded nature of the adapter may provide a 3'-hydroxyl group from which second-strand synthesis can be initiated, thus preventing concatamerization of the adapter during ligation, since T4 RNA ligase cannot ligate double-stranded nucleic acids.

(E) Tearing off the carrier / rewinding

In some studies, a vector was ligated to cDNA that had been synthesized on a tip. The cDNA or vector:cDNA was cleaved from the solid support with AscI in a volume of 40 μΐ at 37°C on a rotating rack in a hybridization oven for 4 hours. The cleaved DNA was then heated at 70°C for 20 minutes with or without agitation. The vector:cDNA was directly recirculated by adding ligation buffer and T4 DNA ligase to make up the volume to 50 μΐ. The cDNA that had not previously been ligated to the vector was divided into several aliquots. From these aliquots, tests were performed to determine which vector:insert ratio gave the best results in subsequent transformation. Since the total amount of cDNA is small (50-120 ng in a 40 μΐ volume), the entire ligation mixture was transformed, necessitating some desalting step.

Within four hours, more than 90% of the cDNA can be cleaved from the tip. Under these conditions, there is no benefit to increasing the incubation time. Although the cleavage time can be reduced by increasing the enzyme concentration, the incubation time and the cleavage volume must be in balance.

(F) Transformation

The cDNA clones were amplified in electrocompetent E. coli cells transformed with a DNA sample from the ligation - by electroporation. In the transformation, DNA concentrations varied between 1O ⁹ -1O ¹⁰ cfu/pg. It is well known to the skilled person that the main limitation of this process is the salt sensitivity of electroporation. Only 1-2 μΐ volumes (5-10% of the total volume) of a general ligation mixture can be electroporated per electrocompetent cell sample without exceeding the salt tolerance threshold and the electrical discharge between the electrodes leads to the loss of the ligation mixture by evaporating the cells. It is therefore worthwhile to electroporate several separate cell samples separately, but this requires a lot of laboratory work and is expensive. A better solution is to introduce a desalting step into the present cDNA methodology that is flexible enough to be implemented in an “in-line” or “scale-down” version of the technology without compromising the yield.

Transformants are plated on commonly used growth media, such as LB agar containing antibiotics. Only bacteria that carry the antibiotic resistance gene to the antibiotic present in the medium will form colonies. Separation of true recombinants from colonies carrying only vector sequences is often accomplished by blue-white color selection, in which the blue color is the result of expression of the lacZ gene, which spans the multiple cloning site of most common plasmid vectors. If expression of the product encoded by the lacZ gene is disrupted by an insert ligated to the multiple cloning site, this results in a white colony phenotype. In this scenario, recombinants can be visually distinguished from non-recombinants, but there may be some background and inadvertent spike-in non-recombinants in the final product. Even a relatively low background of non-recombinants can cause problems when the library is expanded, as bacteria containing vectors without an insert grow faster and are much more stable than recombinants containing an insert, especially a large insert.

Example 2: “Scaling down RNA input for cDNA synthesis on solid support

To minimize the amount of material lost during the many manipulations required to prepare a general cDNA library, the RNA input level can be reduced by a factor of 10 to 100. For example, roughly 100 ng of double-stranded cDNA on a solid support can be synthesized using 10 µg of total RNA, which is 50 times less starting material than is required for a commercially available kit. It is possible to reduce this further, by a factor of 10, without significantly altering the recipe described above, but this requires an additional amplification step.

Another option is to perform cDNA synthesis via adapter ligation with a T7 promoter adapter, which allows amplification by in vitro transcription rather than PCR. This procedure eliminates the length bias associated with PCR amplification, allowing for a more representative library. The in vitro synthesized transcripts can then be captured and processed like any other RNA source. To obtain full-length transcripts, the success of amplification relies on careful optimization of the in vitro transcription conditions. Uncaptured transcripts can be polyadenylated with yeast poly(A) polymerase and then re-captured on the same substrate. Although the re-adenylated transcripts are unlikely to be full-length, the information they contain will not be lost from the RNA pool.

In one study, a T7 promoter adapter was synthesized on a mount and ligated to double-stranded cDNA transcribed from 10 pg of RNA. During in vitro transcription, following the manufacturer's recommended conditions, significant amounts of transcripts ranging in size from 300 to 2000 bp were produced. Although these are not optimal sizes for an efficient amplification step, the study demonstrated that it is possible to produce in vitro transcripts on solid supports.

Another strategy for the “scale down” approach is based on asymmetric (linear) PCR, in which the product is amplified and captured in a manner similar to in vitro transcription. Since the amplified product is more like DNA, DNA polymerase is used to produce double-stranded cDNA.

Example 3: Solid support-based assays

Ten pg of poly(A) + mRNA from total RNA was captured, reverse transcribed, second-strand cDNA was synthesized, and a T7 promoter adapter was ligated to the cDNA. In one study, the solid support was placed in a standard Cetus PCR tube (ABI, Foster City, CA) and run for 35 cycles with the addition of long PCR polymerase (Ex-Taq, TAKARA). The duration of the 70°C extension step was increased by 1 min every five cycles. The single primer used in the PCR was complementary to the adapter, so that amplification started from the 5' end of the ligated cDNA, producing many (+) strand copies. After the cycles, the PCR products were heat-denatured and run on an agarose gel to visualize the length range of the single-stranded cDNA. The size range was roughly from 500 bp to over 20 kilobase pairs, which corresponded well to the results obtained from the labeled double-stranded cDNA cleaved, electrophoresed, and visualized by autoradiography in the control assay run in parallel.

To determine whether the single-stranded PCR product represents the original mRNA population, we designed PCR primers for several genes known to be expressed at high, low, or absent levels. We designed primers such that all products were of approximately the same length (400-600 bp) and located close to the 5' end of the cDNA. . This primer design provided a good estimate of the quality of first-strand synthesis. Since the RNA source was a human T-cell line, the primers used were IL-2, IL-4, GM-CSF, GAPDH, CTLA4, c-fos, and Werner helicase sequences. Murine guanylate kinase was used as a negative control.

As expected, all polymerase chain reactions yielded products of the expected size, except for CTLA4 and murine guanylate kinase. The product obtained in the case of IL-2 was confirmed by Northern blot. 50 ng of putative IL-2 was labeled with ³² P and hybridized to a blot containing immobilized RNA from stimulated (PMA+ionomycin) and unstimulated Jurkat. After very stringent washing, the unstimulated RNA showed virtually no signal, while the stimulated sample gave an intense signal in the size range corresponding to IL-2.

Example 4: Application of solid support for rapid amplification of cDNA ends

Rapid amplification of cDNA ends (RACE), a technique that can also be adapted to the solid-support-based cDNA technology described herein. Either 5'- or 3'-RACE can be performed after ligation of an adapter to double-stranded cDNA, using either an anchoring oligonucleotide (3'-RACE) or the appropriate 5' adapter (5'-RACE). Solid-support RACE has several advantages over currently available techniques, such as the use of fewer materials in the production of cDNA and the reusability of the product. This application is based on the fact that a PCR reaction can be run directly on the solid support described above. Initial experiments have shown that this can be done on a high copy number "housekeeping" gene, GAPDH, and that the final product is directly proportional to the quality of the captured RNA and the subsequently synthesized cDNA. Methods for performing 3'-RACE and 5'-RACE are well known to those skilled in the art [see, for example, Wu et al., "Methods in gene biotechnology," 15-28, CRC Press (1997)].

Example 5: cDNA synthesis on solid support for gene expression studies (A) Cell stimulation and RNA preparation

In a series of experiments, Jurkat JRT 3.5 cell lines were stimulated for 6 hours in serum-free RPMI medium (Life technologies, Gaitersburg, MD) in the presence of 10 mg/ml phorbol-12myristate-13-acetate (Calbiochem, San Diego, CA) and 100 ng/ml ionomycin (Calbiochem) to a concentration of 1*10 ⁶ cells/ml. Cells were pelleted, washed once in PBS (Life Technologies), re-seeded and lysed in 0.5 ml/10 ⁶ cells in buffer (4 M guanidine isothiocyanate, 1% N-lauryl sarcosine, 25 mM sodium citrate, (pH 7.1)) (Fisher Scientific, Pittsburg, PA). One-tenth volume of 2 M sodium acetate (pH 4.2) (Fisher Scientific) was added and then mixed with one volume of water-saturated phenol (Amresco, Solon, OH). One-quarter volume of chloroform:isoamyl alcohol (29:1) was mixed vigorously and incubated on ice for 10 min. The lysate was centrifuged, the aqueous phase was removed, and extracted with an equal volume of chloroform:isoamyl alcohol. The aqueous phase was collected and the RNA was precipitated with 2 volumes of ethanol (Quantum Chemical Corp., Tuscola, IL). After centrifugation, the ethanol was decanted and the RNA was air-dried slightly and then suspended in RNase-free water at a concentration range of 1-5 mg/ml.

(B) Capture and first strand synthesis

A solid support coated with a covalently bound oligonucleotide with the sequence 5'ACTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTT-3' (sequence given as ID No. 1 in the Sequence Listing) (Genset, LaJolla, CA) was added to 10 pg of total cellular RNA and diluted with an appropriate amount of RNase-free water to coat the tip in a 1.5 ml microfuge tube (Fisher Scientific). This oligonucleotide contains a spacer and an Ascl cleavage site at the 5' end of the oligo(dT) sequence. The RNA and tip were incubated for 5 min at 65°C. To each tube, an equal volume of 2* mRNA hybridization buffer (50 mM Tris, pH 7.5, 1 M NaCl) (Fisher Scientific) and 20 pg/ml acetylated BSA (New England Biolabs, Beverly, MA) was added and the tubes were gently shaken at room temperature for 2 min. The supernatant was removed and the tip was washed three times in 1* mRNA hybridization buffer. After the final wash, reverse transcriptase mix (1* MMLV-reverse transcriptase buffer, 1 mM dNTP mix, 2 mM DTT (Life

- 67 Technologies), 20 units of Rnasin (Promega, Madison, WI), 10 pg/ml of acetylated-BSA (New England Biolabs) were added, and 600 units of MMLV reverse transcriptase (Life Technologies) were added. The reaction mixture was incubated at 42°C for 2 hours with gentle shaking. Then, one unit of RNase H (Boehringer-Mannheim, Indianapolis, IN) was added and the reaction was continued for another half hour. The supernatant was removed again and each tip was washed three times in 10 mM Tris (pH 8, + 1 mM EDTA) (Fisher

The remaining RNA template was removed by rinsing the tips in TE + 0.01% SDS buffer (Fisher Scientific).

Scientific) boiled.

Example 6: High-performance melting process using a solid support

A capture oligonucleotide (36-mer) was covalently attached to a poly(ethyleneimine)-coated nylon tip via the C6-amine site. In a 1.5 M guanidinium thiocyanate solution, shorter oligonucleotides (18-mer) labeled with Texas Red at the C6-amino site were hybridized to the capture oligonucleotide for 15 min at room temperature. The tips were then washed twice in TEN buffer (0.01 M Tris, pH 7.5, 1 mM EDTA, 100 mM NaCl) and once in TENS buffer (0.01 M Tris, pH 7.5, 1 mM EDTA. 100 mM NaCl, 0.1% SDS), followed by two washes in TEN buffer to remove unhybridized labeled oligonucleotides. Samples from the solutions to be tested were placed in a polycarbonate thermowell plate

(polycarbonate thermowell plate) (Corning Costar Corp., Cambridge, MA) and the plate was placed in a MJ thermal cycler (MJ Research Company, Watertown, MA). The tips were serially transferred from one well of the plate to the next. The temperature was increased by 5°C every 5 minutes, i.e., the starting temperature was 10°C and the final temperature was 85°C. The liquid was then transferred to a black 96-well microtiter plate and fluorescence was measured.

The level of fluorescence measured in each well is related to the amount of labeled oligonucleotide annealed from the capture oligonucleotide. The "de-annealing" or duplex dissociation was performed in the temperature range of 10-95°C. To calculate Td, the accumulated values measured at each temperature were plotted as a function of temperature. The temperature at which 50% of the material dissociated from the tip was considered _Td . The "helical-disordered transition" was considered to be the temperature range from the temperature at which a for a given oligonucleotide duplex (or nucleic acid duplex with or without mismatches in the duplex) is equal to 0.2 to the temperature at which a for the same given oligonucleotide duplex (or nucleic acid duplex with or without mismatches in the duplex) is equal to 0.8. The data were tabulated and the de-annealing curves were calculated for each solution. From these de-annealing curves, the _Td , the aHCT, and AT _d .

In one study, using the 1*12 tip array, 100 μΐ aliquots of each test solution were placed into two thermowell plates (Cornig Costar, Cambridge, MA) containing 16 separate wells (one tube per temperature point). Before each temperature step, the tip array was transferred to a new row of the plate. Just before reaching 50°C, the first plate was removed from the thermocycler and replaced with the second thermowell plate. When the thermocycling program was complete, the liquids from the wells were transferred to two black microfluor plates (Dynatech). Fluorescence was measured at 584 excitation and 612 emission wavelengths. The data were entered into a spreadsheet program and analyzed.

In one study, using the 4*12 tip configuration, 8 thermowell plates were cut in half to create a 4*12-well plate. 100 μΐ of each test solution was measured into one well of each half-plate until each well contained test solution. All 16 half-plates were identical in terms of solution composition. Before each temperature jump, the tip was transferred to a new row of the plate. When the thermocycling program was completed, the liquids from the wells were transferred to two black microfluor plates (Dynatech). Fluorescence was measured at 584 excitation and 612 emission wavelengths. The data were entered into a spreadsheet program and analyzed.

Example 7: Determination of the melting temperatures of oligonucleotide duplexes in different hibotropic and non-hibotropic hybridization solutions

In this example, we describe the determination of the T _d of wild-type and mutant oligonucleotides upon hybridization to a target nucleic acid. We demonstrate that the hibotropic-based hybridization solution allows the detection of a single base pair mutation in the target nucleic acid with a probe of up to 30 nucleotides in length.

(A) Solutions and reagents

The filter washing solution (FW) was 0.09 M NaCl, 540 mM Tris (pH 7.6), 25 mM EDTA. The “SDS/FW solution” was a FW solution containing 0.1% sodium dodecyl sulfate. The hybridization solution contained the concentration of hibotropic (2% N-lauryl sarcosine (sarcosyl)) specified in the text, 50 mM Tris (pH 7.6), and 25 mM EDTA. The formamide hybridization solution contained 30% formamide, 0.09 M NaCl, 40 mM Tris-HCl (pH 7.6), 5 mM EDTA, and 0.1% SDS. GuCl, lithium hydroxide, trichloroacetic acid, NaSCN, NaClO ₄ , and ΚΙ were obtained from Sigma (St. Louis, MO). Rubidium hydroxide was purchased from CFS Chemicals (Columbus, OH). CsTFA was obtained from Pharmacia (Piscataway, NJ).

LiTCA, TMATCA and TEATCA were prepared by titrating appropriate 3 N LiOH, TEAOH and TMAOH solutions with trichloroacetic acid (100% W/v, 6.1 N) dropwise on ice with continuous stirring to pH 7.0. The salt was dried under vacuum, washed once with ether and then dried.

Oligonucleotides were synthesized on a commercially available synthesizer using standard cyanoethyl-N,N-diisopropylaminophosphoramidite (CED-phosphoramidite) chemistry. Amine tails were incorporated at the 5' end using commercially available N-monomethoxytritylaminohexyloxy-CED-phosphoramidite. In some cases, oligonucleotides were purchased commercially (Midland Certified Fkagents, Midland, Tx).

Table 1 shows the oligonucleotides used to measure the difference in T _d -i between the wild-type oligonucleotide and the mutant oligonucleotide. The wild-type oligonucleotide was a completely and perfectly matched duplex, while the mutant oligonucleotide contained a mismatched base pair (usually in the middle of the oligonucleotide).

Table 1:

Oligonucleotide Nucleotide sequence Secretary identification number "capture oligonucleotide" 5'-GTCATACTCCTGCTTGCTGATCCACATCTG-3' 2 wild type 30-mer 5'-CAGATGGGTATCAGCAAGCAGGAGTATGAC-3' 3 mutant 30-mer wild type 24-mer 5'-CAGATGGGTATCAGGAAGCAGGAGTATGAC-3' 4 5'-ATGGGTATCAGCAAGCAGGAGTAT-3' 5 mutant 24-mer 5'-ATGGGTATCAGGAAGCAGGAGTAT-3' 6 wild type 18-mer 5'-GGTATCAGCAAGCAGGAG-3' 7 mutant 18-mer 5'-GGTATCAGGAAGCAGGAG-3' 8

The oligonucleotides were attached to the tips as described herein. In these studies, the oligonucleotides were attached to the tips as described by Van Ness et al. [Van Ness et al., Nucleic Acid Res., 19, 3345 (1991)]. The oligonucleotide tips contained 0.1-1.2 pg/tip of covalently immobilized oligonucleotide.

(B) Solid-phase hybridization

The oligonucleotide probes were reacted with amine-reactive fluorochromes. The resulting oligonucleotide preparation was divided into three parts and the parts were reacted with either (a) a 20-fold molar excess of Texas Red sulfonyl chloride (Molecular Probes, Eugene, OR), (b) a 20-fold molar excess of Lissamine sulfonyl chloride (Molecular Probes, Eugene, OR), or (c) a 20-fold molar excess of fluorescein isothiocyanate. Unreacted fluorochromes were removed by gel filtration chromatography on a Sephadex G25 column.

We have developed a high-throughput method for measuring the thermodynamic properties of oligonucleotide duplexes. The method can be used to screen thousands of solution samples for their ability to modulate the thermodynamic parameters of the “helical-disordered” transition of oligonucleotide duplexes. The method requires a solid support designed to fit into a Cetus plate (or a well of a plate designed for 96-well PCR format) and a volume of approximately 40 μΐ to be completely covered by the liquid. The tip design is shown in Figure 1. This tip was also designed to fit the square end of a spring probe, which can be used as a mounting end for nylon tips in 1*8, 1*12, 4*8, 4*12, or 8*12 configurations. A drawing of such a device is shown in Figure 2.

One member of the oligonucleotides was immobilized on the nylon tip as described by Van Ness and Chen [Van Ness and Chen, Nucleic Acid Res. 19, 5143 (1991)]. An oligonucleotide duplex was then formed on the tip by a hybridization step. The hybridization step can be performed en masse in a single well or separately in the wells of the PCR plate. Therefore, it is possible to produce different oligonucleotide duplexes with each tip of a 96-member tip array.

Following the hybridization step, the tips are washed and placed on a PCR plate in a thermocycler. In the 1*8 or 1*12 format, when the temperature is increased by 5°C, the tips are always transferred to the next set of wells. Typically, the temperatures differ in 5°C increments, and the thawing time for each temperature is 1-5 minutes. For example, the tips in the 1*12 array are placed in row “H” at 10°C. The thermocycler is then programmed to increase the temperature in 16 steps of 5°C at 2-minute intervals. The tip array is moved from row to row 15 seconds before the temperature increases. In this arrangement, 12 solutions can be studied simultaneously in two plates filled with the solution. In the 96-tip array, the entire plate containing the solution is replaced in the thermocycler at the specified time.

Fluorescent probes are usually used in this setup. Fluorescent probes have only a small influence on the measured T _d values described here. Radiolabeled or fluorescent probes allow the measurement of a wide range of solutions, since, unlike the melting curves determined by UV spectrometry (hyperchromicity shift), there is no prescribed optical purity. Fluorescence is measured in a microtiter plate fluorescence reader and the data is transferred directly to a spreadsheet program, such as Excel, which then calculates the stability, enthalpy, helical-disordered transition temperature range, and then plots the results in a graph. Typically, a study with a l*12 setup, which can measure 12 solutions simultaneously, can be completed within 1 hour, including preparation and data derivation.

In the case of the oligonucleotide-containing tip, to determine the oligonucleotide/oligonucleotide T _d , fluorescently labeled oligonucleotide in different hybridization solutions was incubated with complementary oligonucleotide immobilized on the oligonucleotide tip. At different temperatures (19-65°C), for 5-30 minutes, 5-5000 ng of oligonucleotide was hybridized in a volume of 300-400 μΐ. The tips were then washed first three times with 1-1 ml of the appropriate hybridization solution, and then once - at the starting temperature of the thawing process - with the appropriate thawing solution. The tips in 100-100 μΐ of the appropriate thawing solution were placed in the thermocycler. The temperature was increased in intervals of 1-5 minutes and the tips were moved to a new well of the microtiter plate. The thawing, or duplex dissociation, was performed in the temperature range of 10-95°C. Fluorescence was measured with a commercially available fluorescence plate reader.

To calculate AT _d , the cumulative relative fluorescence units (RFU) released at each temperature tested were plotted as a function of temperature. AT _d or T _m is the temperature at which 50% of the material is dissociated from the tip. By definition, the helical-disordered transition occurs from the temperature at which a is equal to 0.2 for a given oligonucleotide duplex (or nucleic acid duplex, which may or may not contain a base pairing mismatch anywhere within the duplex) to the temperature at which a is equal to 0.8 for the same given oligonucleotide duplex (or nucleic acid duplex).

The following T _d were measured during the hybridizations described below.

Table 2

Type of solution Length of the trial T _d (mutant) (C°) T _d (wild type) (C°) Δ-Td (C°) HCT (C°) 2.5m LiTCA 30-mer 27 33 6 13/14 2.5m LiTCA 24-mer 25.5 32 6.5 13/14.5 2.5m LiTCA 18-mer 24 31 7 9/14 2.0 m LiTCA 30-mer 42 47 5 13.5/16 2.0 m LiTCA 24-mer 38 44 6 14/15 2.0 m LiTCA 18-mer 37 43 6 14.5/16.5 3.0 m GuSCN 30-mer 37 42.5 5.5 13.5/17.5 3.0 m GuSCN 24-mer 34.5 41 12.5/17 3.0 m GuSCN 18-mer 33.5 40.5 7 14.5/15 3.0 m GuHCl 30-mer 55.5 60 4.5 16/21 3.0 m GuHCl 24-mer 52.5 58 5.5 15/20 3.0 m GuHCl 18-mer 50 57 7 18/20 Rapid Hybe 30-mer 80 80 0 no data* Rapid Hybe 24-mer 80 80 0 no data Rapid Hybe 18-mer 68 70 2 18/23 5xSSC 30-mer 72.5 72.5 0 18/18 5xSSC 24-mer 69 70 1 18/18 5xSSC 18-mer 67 72 5 16/18 Promega QY 30-mer 80 80 0 no data Promega QY 24-mer 80 80 0 no data Promega QY 18-mer 62 65 3 20/23

*no data: means it is not appropriate or too long to be accurately determined

The data indicate that the hibotropic solutions (LiTCA, GuSCN and GuHCl) allow the detection of a single base mismatch in a 24-mer and 30-mer probe, whereas the detection of a single base mismatch in standard hybridization solutions (Rapid Hybe, Promega QY or 5* SSC) was not possible.

A similar experiment was performed with 24-mers, using the hybridization solution series described above.

Table 3

Type of hybridization solution slope ([··], k) HCT AT _d LiTCA, 3 M________. 19 8C 7.5 C GuSCN, 3 M 13 10 6.0 NaSCN, 3 M ___________ 8.5 11 5.5 National, 3 M__ 7 12 4.5 OFF, 3 M 5 15 3.0 NaCl, 0.165 M 4.5 17.5 15 GuCl, 3 M 3.5 18 1.2 CsTFA, 2M 2.5 18 1.2 30% formamide ND 20 1.5

AT _d (wt) is the T _d of the perfectly matched oligonucleotide duplex, and T _m (mt) is the T _d of the oligonucleotide duplex containing a single mismatch. The values shown are for the 24-mer sequence duplex described above. From the data presented in Table 3, the stringency factor is directly proportional to the difference between the perfectly matched duplex and the duplex containing the mismatch. That is, the stringency factor predicts the ability of a given hybridization solution to discriminate between duplexes containing mismatches.

Although the foregoing relates to a specific, preferred embodiment of the invention, it should be understood that the invention is not so limited. It will be clear to those skilled in the art that the disclosed solution may be modified, but such modifications fall within the scope of the invention as defined by the following patent claims.

SEQUENTIALIST (210) 1 (211) 39 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

Oligonucleotide used in gene expression studies for capture and first-strand synthesis (400) 1 actactgatc aggcgcgcct ttttttttu tttttttt 39 (210) 2 (211) 30 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 2 gtcatactcc tgcttgctga tccacatctg 30 (210) 3 (211) 30 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 3 cagatgggta tcagcaagca ggagtatgac 30 (210) 4 (211) 30 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 4 cagatgggta tcaggaagca ggagtatgac 30 (210) 5 (211) 24 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 5 atgggtatca gcaagcagga gtat 24 (210) 6 (211) 24 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 6 atgggtatca ggaagcagga gtat (210) 7 (211) 18 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 7 ggtatcagca agcaggag 18 (210) 8 (211) 18 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

For wild-type and mutant oligonucleotides, oligonucleotides used to measure the difference in temperatures at which the signal of nucleic acid duplex molecules is single-stranded.

(400) 8 ggtatcagga agcacjgag (210) 9 (211) 30 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

Reference oligonucleotide representing a nucleic acid duplex in immobilized form (400) 9 gtcatactcc tgcttgctga tccacatctg 30 (210) 10 (211) 24 (212) DNA (213) Artificial sequence (220) (223) Description of the artificial sequence:

Reference oligonucleotide representing a nucleic acid duplex in dissolved form (400) 10 tgtggatcag caagcaggag tatg

Claims (70)

PATENT CLAIMS 1. A solid-phase sample retention tip for synthesizing or detecting nucleic acids, characterized in that it comprises: - a tip configuration that can be connected to a support pin; and - a chemical layer covering at least a portion of the tip formation and suitable for biomolecules to bind thereto, thereby forming a solid-phase biomolecule pattern on the tip formation. 2. The sample retention tip of claim 1, wherein the tip configuration is removably connectable to the holding pin. 3. The sample retention tip of claim 1, wherein the chemical layer is applied directly to the tip design. 4. The needle retention tip of claim 1, wherein the tip configuration is partially conical and includes more than one formed groove. 5. The sample retention tip of claim 1, wherein the tip configuration includes more than one heat exchange fin. 6. The sample retention tip of claim 5, characterized in that the tip configuration has a partially conical shape. 7. The sample retention tip of claim 1, wherein the tip assembly has a socket adapted to receive the holding pin, the socket having a closed end portion and an open end portion, the open end portion being generally funnel-shaped with a cross-section tapering toward the closed end portion. 8. The sample retention tip of claim 1, wherein the tip is made of nylon 6/6. 9. The sample retention tip of claim 1, wherein the chemical layer is a polymer containing more than one amino group, which amino groups can bind to biomolecules. 10. The sample retention tip of claim 1, wherein the chemical layer is a poly(ethyleneimine) layer. 11. The sample retention tip of claim 1, wherein the selected chemical layer is covalently bonded to the tip structure. 12. A solid-phase sample retention structure for synthesizing or detecting nucleic acids, characterized in that it comprises: - a holding pin; - a tip formation connected to the support pin; and - a chemical layer covering at least a portion of the tip formation and suitable for biomolecules to bind thereto, thereby forming a solid-phase biomolecule pattern on the tip formation. 13. The structure according to claim 12, characterized in that the support pin is a spring-loaded pin. 14. The structure according to claim 12, characterized in that the tip formation is movably connectable to the support body. 15. The structure of claim 12, wherein the tip formation is a nylon 6/6 element. 16. The structure of claim 12, wherein the tip formation is partially conical and includes more than one formed groove. 17. The structure according to claim 12, characterized in that the tip formation has more than one heat exchange fin. 18. The structure of claim 12, wherein the chemical layer is a polymer containing more than one amino group capable of binding to a biomolecule. 19. The structure of claim 12, wherein the chemical layer is a poly(ethyleneimine) layer. 20. The structure of claim 12, wherein the tip formation has a corrugated surface that increases the surface area of the tip formation. 21. The structure of claim 12, wherein the support pin has corner projections and is generally polygonal in cross-section, the tip formation has a socket defined by side walls and is generally circular in cross-section, the corner projections frictionally engage the side walls, thereby retaining the tip formation on the support pin. 22. The device of claim 12, wherein the tip formation has a socket adapted to receive the support mandrel, the socket having a closed end portion and an open end portion, the open end portion being generally funnel-shaped with a cross-section tapering toward the closed end portion. 23. A solid-phase sample retention structure arrangement for synthesizing or detecting nucleic acids, characterized in that it comprises: - a foundation; - more than one support pin connected to the base in a selected arrangement, each support pin having an end portion spaced apart from the base; - more than one tip formation connected to the end portion of the support pins; and - a chemical layer covering at least a portion of the tip formation and suitable for biomolecules to bind thereto, thereby forming a solid-phase biomolecule pattern on the tip formation. 24. Arrangement according to claim 23, characterized in that the holding pin is a spring pin. 25. The arrangement of claim 23, wherein the tip formations are removably connected to the support mandrel. 26. The arrangement of claim 23, wherein the tip formations are nylon 6/6 elements. 27. An arrangement according to claim 23, characterized in that the tip formations are partially conical and have more than one groove formed therein. 28. The arrangement according to claim 23, characterized in that each tip formation has more than one heat exchange fin. 29. The arrangement of claim 23, wherein the chemical layer is a polymer containing more than one amino group. 30. The arrangement of claim 23, wherein the chemical layer is a poly(ethyleneimine) layer. 31. The arrangement of claim 23, wherein the tip formation has a corrugated surface. 32. The arrangement of claim 23, wherein each tip configuration has a socket to which the end portion of the corresponding support mandrel is movably connected, the socket having a closed end portion and an open end portion, the open end portion being generally funnel-shaped with a cross-section tapering toward the closed end portion. 33. A combination of a solid-phase sample retention structure and a microtiter plate for use in a method for synthesizing or detecting nucleic acids, characterized in that it comprises: - a microtiter plate, the wells of which are designed to accommodate a volume of a sample containing a biomolecule; and - a solid-phase sample retention structure, which is dimensioned so that at least a portion thereof extends into the hole, and which solid-phase sample retention structure comprises: - a holding pin; - a tip formation associated with the support mandrel, said tip formation being movably positionable within the hole; and - a chemical layer covering at least a portion of the tip formation and suitable for biomolecules to bind thereto, thereby forming a solid-phase biomolecule pattern on the tip formation. 34. The combination according to claim 33, characterized in that the holding pin is a spring-loaded pin. 35. The combination of claim 33, wherein the tip is removably attachable to the mandrel and positioned in the hole when the tip is removed from the mandrel. 36. The combination of claim 33, wherein the tip is a nylon 6/6 element. 37. The combination according to claim 33, characterized in that the tip formation has a cross-sectional shape that fully corresponds to the cross-sectional shape of the hole. 38. The combination of claim 33, wherein the tip is partially conical with a plurality of grooves formed therein. 39. The combination of claim 33, wherein the chemical layer is a polymer containing more than one amino group. 40. The combination of claim 33, wherein the chemical layer is a poly(ethyleneimine) layer. 41. The combination of claim 33, wherein the tip configuration is frictionally retained by the holding mandrel. 42. The combination of claim 33, wherein the microtiter plate has a plurality of one or more wells and further comprises more than one holding pin arranged in a selected arrangement and more than one tip configuration coupled to the holding pin, thereby forming a solid phase sample retaining tip configuration that can be positioned in the wells. 43. The combination of claim 42, characterized in that it comprises a base connected to the end of the support pins remote from the tip formations, and the tip formations are located in substantially the same plane. 44. A combination of a solid-phase sample retention tip and a microtiter plate for use in methods for synthesizing or detecting nucleic acids, characterized in that it comprises: - a microtiter plate, the well of which is designed to accommodate a volume of a sample containing biomolecules; and - a solid-phase sample retention tip that is movably positionable within the well, the tip having a tip configuration that can be engaged with the holding pin while the tip configuration is in the well; and a chemical layer that coats at least a portion of the tip configuration and that is capable of being bound by biomolecules, thereby forming a solid-phase biomolecule pattern on the tip configuration. 45. The combination of claim 44, wherein the tip formation is a nylon 6/6 element. 46. The combination according to claim 44, characterized in that the tip formation has a cross-sectional shape that fully corresponds to the cross-sectional shape of the hole. 47. The combination of claim 44, wherein the tip configuration is partially conical. 48. The combination of claim 44, wherein the tip formation is partially conical and includes more than one formed groove. 49. The combination of claim 44, wherein the chemical layer is a poly(ethyleneimine) layer. 50. A method for producing a solid-phase sample retention tip for use in solid-phase molecular biology procedures, characterized in that a tip configuration connectable to the holding mandrel is formed from a base material; coating at least a portion of the tip structure with a chemical layer, the chemical layer being capable of being bound by biomolecules, thereby forming a solid-phase biomolecule pattern on the tip structure; and attaching the chemical layer to the substrate. 51. The method according to claim 50, characterized in that during the attachment of a chemical layer to the base material, the chemical layer is attached to the base material in a covalent manner. 52. The method according to claim 50, characterized in that the chemical layer comprises a polymer containing more than one amino group, which amino groups are capable of binding to biomolecules. 53. The method of claim 50, wherein the chemical layer is a poly(ethyleneimine) layer and the base material is nylon 6/6, and the fixing step covalently fixes the poly(ethyleneimine) to the nylon 6/6. 54. The method of claim 50, further comprising the step of securing the tip assembly to the support mandrel. 55. The method of claim 50, characterized in that during the attachment of the tip formation, the tip formation is movably attached to the end of the support mandrel. 56. The method of claim 50, further comprising the steps of providing a base, more than one support pin, and more than one tip configuration with the chemical layer thereon, and attaching the support pins to the base in a predetermined order, attaching the tip configurations to a corresponding support pin of the plurality of support pins, thereby forming an array of solid-phase sample retention tips that are spatially separated from the base. 57. A method for converting a biomolecule into a solid-phase sample, characterized in that - a portion of the tip structure - which tip structure has a substrate portion and a chemical layer on the substrate portion and the chemical layer is bindable to biomolecules - is immersed in a solution containing a biomolecule; - enabling the biomolecule to bind to the chemical layer, which biomolecules thus form a solid phase pattern on the tip structure; and - the tip structure is removed from the solution after the biomolecule has already bound to the chemical layer. 58. The method of claim 57, wherein the dipping comprises positioning a portion of the tip structure in a well of a microtiter plate containing the solution. 59. The method of claim 57, characterized in that after the biomolecule has been bound to the chemical layer, a molecular biological process is performed on the tip structure. 60. The method of claim 57, wherein after the biomolecule has been bound to the chemical layer, the solid-phase tip structure is stored in the storage component. 61. The method of claim 60, wherein the storage component is a microtiter plate having holes therein, and the storage step comprises placing the tip structure in the hole after the biomolecule has bound to the chemical layer, and placing the microtiter plate and the tip structure as a unit in the storage location. 62. The method according to claim 57, characterized in that the biomolecule used is a nucleic acid or an amino acid polymer. « · » ** ♦ « Ο V · * ♦ · “:· ··:· /. 63. The method according to claim 62, characterized in that the nucleic acid used is an oligonucleotide having one end bound to said chemical layer and the other end free. 64. The method according to claim 63, characterized in that the oligonucleotide used is an oligonucleotide containing an oligo(dT) sequence at its free end. 65. The method of claim 64, wherein poly(A+)RNA is attached to the tip structure, wherein the poly(A+) portion of the poly(A+)RNA is attached to the oligo(dT) sequence of the oligonucleotide. 66. The method of claim 65, characterized in that cDNA is synthesized from the bound poly(A+)RNA. 67. The method according to claim 57, characterized in that the biomolecule used is an avidin molecule. 68. The method of claim 67, further comprising the step of attaching an oligonucleotide containing at least one biotin moiety and bound to the avidin molecule to the tip structure. 69. The method according to claim 59, characterized in that any of the following is performed as a molecular biological method: cDNA synthesis, polymerase chain reaction, preparation of a subtractive cDNA library, synthesis of various probes, solid-phase minisequencing, oligonucleotide ligation assay, amplified fragment length polymorphism analysis. 70. The method of claim 62, wherein the amino acid polymer is an antibody. The authorized person: Danube Patent and Trademark Office Ltd. Adam Swinger