MXPA00006498A - Solid-phase tips and uses relating thereto - Google Patents

Abstract

Solid-phase assays have provided a powerful approach to the analysis of biomolecules in medical diagnosis and in basic research. Solid-phase nucleic acid hybridization methods, for example, have been applied to analysis of genetic polymorphisms, diagnosis of genetic disease, cancer diagnosis, detection of viral and microbial pathogens, screening of clones, and ordering of genomic fragments. A new solid-phase sample-retaining tip provides improved procedures for synthesizing or detecting a biomolecule. These tips can be used to devise sample-retaining assemblies, which in turn, can be combined to form arrays of solid-phase sample-retaining assemblies useful in automated processes. The tips may be connectable to a spring biased support pin and also contain a chemical layer coating said tip to which a biomolecule is bindable.

Description

SOLID PHASE TIPS AND USES RELATED TO THE SAME TECHNICAL FIELD The present invention relates generally to the application of solid phase techniques for synthesizing and analyzing nucleic acid molecules. In particular, the present invention relates to solid phase supports that can be used to perform a variety of biomolecular procedures.

BACKGROUND OF THE INVENTION Nucleic acid hybridization provides specific character for the recognition of biomolecules, such as DNA and RNA sequences, and has become a powerful technique in medical diagnosis. For example, nucleic acid hybridization methods have been applied for the analysis of genetic polymorphisms, diagnoses of genetic disease, diagnosis of cancer, detection of viral and microbial pathogens, classification of clones and arrangement of genomic fragments (for a review, see Chetverin and Kramer, Bio / Technology 12: 1093, 1994) , the development of automatic synthesis of oligonucleotide probes has also promoted the development of rapid, simple and inexpensive diagnostic assays based on nucleic acid hybridization. The use of DNA probes in analytical techniques has been reviewed by Matthews and Kricka, Anal. Biochem. 169: 1, 1988 (also see Keller and Mank (eds.), DNA Probes, 2nd Edition (Stockton Press 1993), Persing et al., Diagnostic Molecular Microbiology (American Society for Microbiology 1993). A general aspect in nucleic acid hybridization requires immobilization of the target nucleic acid on solid supports, such as nitrocellulose filters and nylon membranes, followed by hybridization with a detectable nucleic acid probe. The disadvantage of such methods is that the immobilized nucleic acid is typically not hermetically bound, resulting in the loss of the target material from the support. In addition, only a small amount of the nucleic acid molecules is available for hybridization. These problems can be overcome through a "sandwich type" hybridization assay wherein the target nucleic acid is hybridized to a "capture" oligonucleotide that has been covalently immobilized to a solid support. Then, a detectably labeled probe is hybridized to a different region of the captured target nucleic acid, and the presence of the probe is measured. The discovery of new therapeutic targets and diagnostic markers has been improved through techniques for analyzing gene expression patterns derived from databases of large expressed sequence tags (Fannon, Trends Biotechnol., 14: 294, 1996). Said sequence data, derived from a wide variety and collections of the cDNA, offer ample information to identify genes for the development of pharmaceutical products. Comparisons of expression patterns from normal and diseased tissues also provide interferences with respect to gene function, and medically identify relevant genes as candidates for therapeutic research and development programs. An important barrier to a more widespread use of solid-phase cDNA synthesis and the use of DNA probes in simple assays has been the lack of solid support and immobilization method that are fully compatible with the hybridization process. The use of solid supports in hybridization based on DNA probe has been reviewed by Meinkoth and Wahl, Anal. Biochem. 138: 267, 1984. Poly (ethyleneimine) ("PEI") has been extensively used in the art to bind biomolecules. PEI is very effective in this capacity for a variety of reasons. For example, PEI is very hydrophilic and thus easily moistens aqueous solutions containing biomolecules. In addition, PEI contains many amino groups, which form salts in acidic groups in a biomolecule. However, the ease with which the PEI accepts aqueous solutions of biomolecules is precisely why, to date, it has not been seen to be used in the preparation of biomolecular provisions. When biomolecular aqueous solutions are placed on a PEI layer the solution is quickly impregnated through the PEI coating instead of remaining at a discrete site. Spring probes are generally well known, as they were introduced earlier in the development of the printed circuit board industry. These are mechanical devices designed to meet the need for precision and reliability in the construction and testing of a variety of electronic components and their connections when assembled in operating circuit boards. Spring probes are essentially electromechanical devices, typically consisting of a tubular housing enclosing a compression spring, ball and plunger. Some probes are specifically designed to carry a flow of electrical current, while others are used to drill, clamp and secure components to a circuit board, and others are designed to perform welding operations. There is nothing in the design or manufacture of spring probes that suggests its potential use as a mechanical device for the transfer and disposal of solutions on solid support for use in the fields of microbiology, biochemistry or molecular biology. Accordingly, there is a need for highly efficient, cost-effective means for arranging or arranging biomolecules on a solid support. The present invention provides these and other related advantages as described in detail hereinafter.

COMPENDIUM OF THE INVENTION The present invention provides a solid phase sample retention assembly that overcomes the disadvantages experienced by the prior art and provides additional related advantages. In one embodiment of this invention, a solid phase sample retention tip is provided that can be used in a method for synthesizing or detecting a biomolecule. The sample holding tip includes a solid support tip structure that can be connected to a support pin, and a chemical layer covers at least a portion of the tip structure. The chemical layer can be attached to the biomolecule to form a solid phase sample of the biomolecule on the tip structure. In one embodiment, the tip structure is removably connected to the support pin. The tip structure has a partially conical shape with a plurality of grooves formed therein. These grooves define a plurality of heat exchange fins that allow the tip structure to heat up and cool rapidly during selected thermal cyclization procedures for the synthesis or detection of biomolecules. In another embodiment of the invention, a sample retention assembly is provided for use in a solid phase process for synthesizing or detecting a biomolecule. The sample retainer assembly includes a support retention, a tip structure connected to the support pin, and a chemical layer covering at least a portion of the tip structure. The chemical layer can be attached to the biomolecule in order to form a solid phase sample of the biomolecule on the tip structure. In one embodiment, the support pin is a spring probe or other spring pin, and the tip structure is a nylon 6/6 member that is removably connected to the spring probe. In another embodiment of the invention, an arrangement of solid phase sample retaining assemblies is provided, wherein a plurality of support pins is connected to a base in a selected arrangement. Each support pin has an end portion that is spaced from the base and a plurality of tip structures are connected to the end portions. The chemical layer covers at least a portion of each tip structure. The chemical layer can be attached to a biomolecule to form a solid phase sample of the biomolecule. In another embodiment of the invention, a solid phase sample retention assembly is combined with a microtiter plate. The microtiter plate has a cavity that is configured to contain a volume of a sample having a biomolecule therein. The solid phase sample retention assembly is sized to extend at least partially into the cavity. The solid phase retention assembly includes a support pin, a tip structure connected to the support pin with the tip structure being removably removable in the cavity, and a chemical layer covering at least a portion of the tip structure . The chemical layer can be attached to the biomolecule and the solution to form a solid phase sample of the biomolecule. In one embodiment of the invention, the microtitre plate of a plurality of cavities therein and the solid phase sample holding assembly includes a plurality of support pins disposed in a selected arrangement, and a plurality of tip structures are connected to the support pins to form a disposition of solid phase sample retaining tips that can be placed in the plurality of cavities. In another aspect of the invention, the solid phase sample retaining tips are combined with a microtiter plate having a plurality of cavities therein. The solid phase sample retaining tips can be removably placed within the cavities of the microtiter plate. The microtitre plate and the sample holding tips can be stored as a unit, so that a solid phase sample on the sample holding tip can be easily stored until it is needed for a synthesis or analysis procedure.

Another aspect of the invention provides a method for manufacturing the solid phase sample retention tip for use in a solid phase molecular biology process. The method includes the steps of forming a substrate material such as a tip structure that can be attached to a support pin, covering at least a portion of the substrate material with a chemical layer that can be attached to selected biomolecules to form a substrate. a sample of solid phase of the biomolecule, and join the chemical layer to the substrate. In one embodiment, the chemical layer is a poly (ethyleneimine) and the substrate material is a nylon 6/6 material, and the step of attaching the chemical layer to the substrate material includes covalently linking the poly (ethyleneimine) to the nylon 6/6. Another embodiment of the invention is directed to a method for forming a solid phase sample of a biomolecule. The method includes the steps of submerging a portion of a tip assembly of a solution having a biomolecule therein. The tip assembly has a portion of substrate and a chemical layer on the substrate portion, with the chemical layer being able to bind to the biomolecule. The biomolecule is allowed to attach to the chemical layer to form a solid phase sample of the biomolecule on the tip assembly, and the tip assembly is removed from the solution after the biomolecule has bound to the chemical layer. In another aspect of the invention, the method includes the step of storing the solid phase tip assembly in a retention member after a biomolecule has been attached to the chemical layer. The retention member in one embodiment of the invention is a microtitre plate with a cavity therein. The step of storing includes placing the tip assembly in the cavity after the biomolecule has been attached to the chemical layer, and placing the microtiter plate and tip assembly as a unit in a storage site.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an isometric view of an arrangement of solid phase sample retaining assemblies according to the illustrative embodiment of the present invention. Figure 2A is an enlarged cross-sectional view of a solid phase retention assembly taken substantially along line 2-2 of Figure 1. Figure 2B is a cross-sectional view of a sample retention assembly of solid phase of an alternative modality. Figure 3 is an enlarged partially cut-away view of a tip structure of a sample retainer assembly of Figure 1. Figure 4 is an enlarged cross-sectional view of the tip structure taken substantially along line 4- 4 of Figure 3. Figure 5 is a side elevational view of the arrangement of Figure 1 shown in solid lines placed above a microtiter plate with a plurality of cavities with the biomolecule samples therein, and shown in schematic lines in a lowered portion with the tip structures placed inside the cavities. Figure 6 is an enlarged side elevational view of the arrangement of Figure 1, shown with a plurality of tip structure positioned in the recesses of a microtiter plate. These and other aspects of the present invention will be apparent upon reference to the following detailed description and accompanying drawings. In addition, several references are identified below and are incorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate the understanding of the invention. A "structural gene" is a nucleotide sequence that is transcribed to a messenger RNA (mRNA), which is then translated into an amino acid sequence characteristic of a specific polypeptide. As used in this"nucleic acid" or "nucleic acid molecule" refers to any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by either ligation , cleavage, endonuclease action and exonuclease action. Nucleic acids can also be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or naturally occurring nucleotide analogues (eg, α-enantiomeric forms of naturally occurring nucleotides), or a combination of both . The modified nucleotides may have modifications in the sugar and / or pyrimidine portions or purine base portions. Sugar modifications include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or the sugars can be functionalized as ethers or esters. In addition, the entire sugar portion can be replaced with spherically and electronically similar structures, such as aza sugars and carbocyclic sugar analogues. Examples of modifications in a base portion include purines and alkylated pyrimidines, acrylated purines or pyrimidines, or other well-known heterocyclic substitutes. The nucleic acid monomers can be linked through phosphodiester linkages or analogs of said linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphorus anilidate, phosphoramidate, and the like. The term "nucleic acid" also includes so-called "peptide nucleic acids", which comprise naturally occurring or modified nucleic acid bases linked to a polyamide base structure. The nucleic acids can be either single chain structure or double chain structure. 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 separated from the genomic DNA of a mammalian cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. In the present invention, the term "biomolecule" refers to either a nucleic acid molecule or a polymer of amino acids or amino acid analogs. As used herein, a "detectable label" or "detectable label" is a molecule or atom, which is conjugated to a nucleic acid molecule to produce a probe that is useful for detection methods. Examples of such labels or labels include photoactive agents or dyes, radioisotopes, fluorescent agents, mass spectrometry labels, and other molecules and label portions. Suitable fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, halofycocyanin, o-phthaldehyde, and fluorescamine. Examples of chemo-luminescent brand compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester. Bioluminescent compounds that are useful for such labels include luciferin, luciferase and aecorin. "Complementary DNA (cDNA)" is a DNA molecule of individual chain structure that is formed from an mRNA template through enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is used for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a DNA molecule of double-stranded structure consisting of said DNA molecule of individual chain structure and its complementary DNA strand structure. The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, the expression involves the transcription of the structural gene of the mRNA and the translation of the mRNA to one or more polypeptides. A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites where foreign nucleotide sequences can be inserted in a determinable manner without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. An "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, gene expression is placed under the control of a promoter, and optionally, under the control of at least one regulatory element. Said gene is said to be "operatively linked to" the promoter. Similarly, a regulatory element and a promoter are operably linked if the regulatory element modulates the activity of the promoter. A "recombinant host" can be any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene in the chromosome or genome of the host cell. As used herein, "hibotrope" refers to any chemical or any mixture of a chemical in an aqueous or organic environment with pH regulators, chelating agents, salts and / or detergents that change the enthalpy of a nucleic acid duplex. at least through 20% when referring to a standard salt 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 oligonucleotide is 5'-GTCATACTCCTGCTTGCTGATCCACATCTG-3 '[SEQ ID NO: 9] as the immobilized oligonucleotide, and 5'TCTGGATCAGCAAGCAGGAGTATG-3' [SEQ ID NO: 10] as the solution nucleotide that is typically labeled at the end 5 'with a fluorochrome such as Texas red. The oligonucleotide duplex (length of 24 nucleotides) has a helical to spiral transition (HCT) of 25 ° C or less. The HCT is the difference between the temperatures at which 80% and 20% of the duplex are of individual chain structure. The average minimum slope for a solution will be defined as a hibotrope that is the first derivative of HCT and is equal to 2.4 in units of 1 / temperature in degrees centigrade ((80% of chain structure indvidual-20% of individual chain) / 25 ° C). As used herein, "Tm" is the temperature at which half of the molecules of a nucleic acid duplex are of individual chain structure. The Tm is measured in solution, while the Td is measured for the duplex fixed in a solid support, both terms indicate the temperature at which half of a duplex is of individual chain structure. As used herein "severe or severe" is the percentage of bases of mismatched pairs that are tolerated for hybridization under a given condition.

As used in the present "discrimination" is the difference in Td between a duplex of perfect base pairs and a duplex containing a mismatch. As used herein, "discrimination temperature" is a temperature at which a hybridization reaction is performed that allows for detectable discrimination between a maladaptive duplex and a perfectly matched duplex. 2. Solid Support A disposition 10 of solid phase sample retaining assemblies 12 according to an illustrative embodiment of the invention is shown in the drawings for illustrative purposes. As best seen in Figure 1, the arrangement 10 includes a plurality of sample retainer assemblies 12 attached to a base structure 14. Each sample retainer assembly 12 includes a support pin 16 securely fixed at one end. to the base 14, and a sample retaining tip structure 20 is attached to the other end 22 of the support pin 16. Each tip structure 10 in the illustrative embodiment is a solid support structure of Nylon 6/6, and the Nylon 6/6 is coated with a layer of poly (ethyleneimine) (PEI) or another selected chemical layer. The PEI layer 24 or other selected chemical layer is adapted to bind to a selected biomolecule to form a solid phase sample that is used in a method for synthesizing or detecting one or more nucleic acids.

The arrangement 10 of the illustrated embodiment includes 8 substantially parallel rows of 12 sample holding assemblies 12 to define an arrangement with 96 sample retaining assemblies equally spaced along the base structure 14. Each sample holding assembly 12 it has approximately the same length so that the tip structures 20 are equally spaced from the base, thus defining a substantially coplanar arrangement of solid phase sample retaining tip structures. The tip structures 20 are spaced apart to match a conventional 96-well Cetus plate or microtiter plate that is adapted to receive and retain selected liquid samples of biomolecules or nucleic acids. Although the illustrative embodiment has an 8 x 12 arrangement of sample retaining assemblies 12, alternative embodiments have other configurations, including a 1 x 8 arrangement, a 1 x 12 arrangement and a 4 x 12 arrangement, as well as provisions larger such as a 16 x 24 arrangement. In the illustrative embodiment, the 96 tip structures 20 are adapted to be immersed in the cavities of the Cetus plate with the biomolecules therein so that the biomolecules chemically bind to the PEI layer 24. When the tip structures 20 are removed from the sample, the biomolecules are adhered to the PEI layer, thus forming the solid phase sample of the biomolecule. The tip structures 20 with the solid phase sample therein can then be used for synthesis or analysis procedures, such as a solid phase nucleic acid assay and detection process as described in more detail below. In one embodiment, the arrangement 10 is installed in a robotic or automatic actuator, so that the base 14 is clamped in the actuator and the sample retaining assemblies 12 project from the base. The actuator quickly and accurately moves the arrangement 10 during the automatic test to selected controlled positions or stations with a predetermined test, synthesis or analysis process. Said automatic test with arrangement 10 and 96 solid phase samples allow substantially faster testing, synthesis or analysis procedures. The arrangement is well suited for such automatic processing, in part, due to the support pins 16 of the sample retaining assemblies 12. As best seen in Figure 2A, each support pin 16 is a spring probe that is typically used for the construction and testing of electronic components, but has been adapted for use in the present invention. The spring probe generally includes a housing 28 enclosing a biasing member 30. A plunger 32 extends toward the housing 28 so that a first end 34 of the plunger is inside the housing 28 adjacent to the biasing member and a second end 36 is outside the accommodation. The deflection member 30 in the illustrative embodiment is a compression spring that pushes axially against the plunger 32 towards the base 14. The first end 34 of the plunger has a shoulder 38 which engages a detent 40 projecting radially inwardly from the housing 28 to limit the maximum extension of the plunger 32 with respect to the housing. The second end 36 of the plunger is fixedly attached to the base 14, and the plunger 32 projects substantially and perpendicularly from the base. In the illustrative embodiment, the housing 28 includes concentric internal and external tubular barrels, 42 and 44, wherein the diverting member 30 and the first end 34 of the plunger are contained within the inner barrel. The external barrel 44 removably receives the inner barrel 42 therein and frictionally engages the inner barrel so that the inner and outer barrels are removably attached to each other. The outer barrel 44 terminates in a distal end portion 46, which is spaced apart from the base 14 and which is connected to the tip structure 20. Accordingly, the outer barrel 44 of the housing and the tip structure 20 are removable as a unit. from the inner barrel 42 and the plunger 32, which remain fixed to the base 14. In this way, an external barrel 44 and the tip structure 20 can be quickly and easily replaced as a unit without having to replace the entire probe of spring. Proper spring probes are available at Everett Charles (Pomona, California), Interconnect Devices, Inc. (Kansas), Test Connections, Inc. (Upland, California), and other manufacturers. Although the illustrative embodiment utilizes spring probes as the support pins 16, other support pins, including deviated and non-diverted support pins, are used in alternative embodiments of the invention. As best seen in Figure 2B, an alternative embodiment of the invention includes the spring probe as the spring pin 16, but the spring probe is oriented 180 ° from the embodiment described above and illustrated in Figure 2A. For example, the remote end portion 46 of the external barrel 44 is fixedly attached to the base 14, and the second end 36 of the plunger 32 is spaced from the base and connected to the tip structure 20.

The spring probes provide a safety aspect that protects the disposition 10 from damage during operation. During a sampling or analysis process, for example, where the arrangement 10 is moved to selected positions and the tip structures 20 are submerged in the cavities of the Cetus plate, or the like, and if the support pins 16 or the Tip structures inadvertently impact with a surface or other object, the spring probe will compress axially to absorb the impact and then return to the uncompressed position. As best seen in Figure 3, the tip structure 20 of the illustrative embodiment has a truncated conical shape with a plurality of channels or slots 50 formed therein. The tip structure can be connected to a support pin, or it can be unitary with the support pin. The slots 50 are V-shaped slots extending axially between a flat distant face 48 and a flat near face 54. The slots 50 have veins or ridges 52 converging from the near face 54 toward the distant face 48 at a selected angle . The truncated conical shape of the tip structure 20 is selected so that it virtually and identically matches the bottom transverse shape of a Cetus plate cavity. Accordingly, the tip structure 20 is configured and dimensioned to be fixed in a very precise position within the cavity of the Cetus plate. The tip structure 20 includes a pin receiving aperture 56 with an open near end 58 on the near face 54 and a closed distant end 60 on the middle portion between the near and distant faces 54 and 58 of the tip structure, respectively . The pin receiving aperture 56 is configured and dimensioned to removably receive the remote end portion 46 of the support pin. Accordingly, the tip structure 20 is removably connected to the support pin 16. However, the tip structure can alternatively be permanently connected to the pin, and in fact, the pin and tip structure can be a single unitary structure. .

In the illustrated embodiment, the pin receiving opening 56 is coaxially aligned with the longitudinal axis of the tip structure. The near opening portion 59 is generally funnel-shaped, so that the open near end 58 of the opening has a diameter larger than the closed distant end 60. The funnel-shaped near portion 59 is adapted to receive the portion distant end 46 of the support pin therein. In the case where the support pin is slightly misaligned relative to the opening 56 during an installation procedure, the near funnel-shaped portion 59 will receive and direct the support pin 16 to a position such that the spring probe is coaxially aligned with the tip structure 20. As best seen in Figure 4, the aperture 56 in the illustrative embodiment is defined by an axial inner wall 61 of the tip structure 20 and has a substantially circular transverse shape. The distal end portion 46 of the spring probe, however, has a substantially square transverse shape with 4 corners 63. The distal end portion 46 of the spring probe is dimensioned such that the corners 63 frictionally engage the interior wall of the spring. tip structure 61 for the purpose of frictionally retaining the tip structure 20 on the support pin 16. In an alternative embodiment of the invention, the end portion has a polygon-shaped cross-sectional area with a plurality of corners that couple the wall interior 61 of the tip structure. As an example, a cross-sectional area with an octagonal shape having the 8 corners frictionally engaging the inner wall 61. In another alternative embodiment, the distal end portion of the support pin has a circular transverse shape that substantially corresponds to the circular cross-sectional area of the aperture. pin reception 56, so that the tip structure 20 is adjusted by pressure on the remote end portion 46 of the spring probe and remains frictionally retained therein. In another embodiment, the tip structure 20 is adhered to the remote end portion 56 with a conventional adhesive so that the tip structure is permanently fixed to the support pin 16. As best seen in Figure 4, the slots 50 and the flanges 52 define the tip structure with truncated conical shape 20 with a generally star-shaped cross-sectional area. As a result, the tip structure 20 has an enlarged outer surface 62, so that a larger amount of biomolecules can be attached to the PEI layer 24 during the formation of the solid phase sample. In the illustrative embodiment, the slots, ridges, distal face 48 and near face 54 of the tip structure 20 define a high surface area, the solid support of Nylon 6/6 which is covalently bonded to the layer of PEI 24. In alternative embodiments, the tip structure 20 is made of a solid substrate, such as glass or silicon, and the PEI layer 24 is covalently bound to the solid substrate using silylation chemistry, as discussed in detail below. In an alternative embodiment, the outer face 62 of the tip structure 20 along the slots 50 and flanges 52 has small holes, in order to provide an additional increased surface area along which the PEI layer 24 will join. In one embodiment, the small holes are usually microscopic, and in an alternative mode, the small holes are macroscopic. Accordingly, the tip structure with small holes 20 provides a larger reaction surface for greater efficiency in the synthesis or analysis procedures. During the selected synthesis or analysis procedures, the tip structure 20 is thermocycled, wherein the tip structure 20 is cycled between high and low temperatures. The ridges 52 of the tip structure 20 form a plurality of heat exchange fins 64 that allow a faster temperature change of the tip structure during the thermal cyclization. As a result, thermocycling can be performed more quickly and more efficiently. As best seen in Figure 5, the arrangement 10 is adapted to be combined and used with a Cetus or microtiter plate 70 having a plurality of cavities 72 therein. As discussed above, the shape of a portion of the cavity 72 substantially coincides with the truncated conical shape of the tip structure 20, consequently, the ridges 52 substantially engage the side walls 74 of the cavity 72 and the flat distal face 48. of the tip structure is placed against the bottom 76 of the cavity. In the preferred embodiment, the microtiter plate 70 has an arrangement of cavities formed by 8 substantially parallel rows of 12 cavities 72 to form the 96-cavity configuration that matches the tip structures of the arrangement 10. In other embodiments, the plates of microtitre 70 have provisions of 1 x 8 cavities, 1 x 12 cavities and 4 x 12 cavities, as well as larger arrangements such as a 16 x 24 cavity format. During the use of the arrangement 10, the arrangement can be automatically or manually moved from an elevated position, shown in solid line in Figure 5 with the tip structures 20 being out of the cavities 72, towards a downward position, shown in dotted lines with the tip structures positioned within the cavities 72. The cavities 72, in one example, contain a liquid sample with the selected biomolecules therein. When the arrangement 10 is in the downward position and the tip structures 20 are in the liquid sample, the chemical reaction occurs between the PEI layer 24 and the biomolecule, in order to form the selected solid phase sample of the biomolecule . In the illustrative embodiment, the cavity 72 has a depth that is approximately 33% greater than the tip structure 20, so that when the tip structure is bathed in the cavity, the liquid sample flows over the entire tip structure to join the most biomolecules possible. As best seen in Figure 6, the arrangement 10 of the sample retaining assemblies 12 can also be used by placing the tip structures 20 within the cavities 72 and separating the tip structures from the support pins 16, as shown in solid lines, so that the tip structures remain in the cavities. The base 14 and the support pins 16 are then moved as a unit away from the microtiter plate 70. As a result, the microtiter plate 70 with the 96 tip structures 20 retained or stored within the cavities 72 can be moved as a unit, and, as an example, being placed in cold storage sites or other suitable storage sites until the solid phase samples are necessary for a selected synthesis or analysis procedure. In the illustrative embodiment, the cavities 72 retain the tip structures 20 at a very precise site relative to the microtiter plate 70, so that the tip structures can be easily and substantially installed simultaneously on the support pins 16. As an example, the microtiter plate 70 is maintained in a known and fixed location, and the base 14 and the support pins 16 move as a unit, either automatically or manually to a selected position above the cavities 72. , so that the support pins substantially and coaxially align with the pin receiving opening 56 in the tip structures. The base 14 and the support pins 16 are then moved towards the microtiter plate 70, so that the support pins 16 are compressed in the openings in the tip structures, thus releasably connecting the tip structures to the support pins . The base 14, the support pins 16 and the tip structures 20 then move as a unit away from the microtiter plate 70, thus removing the tip structures 20 from the cavities 72. The sample retaining tip assemblies 12 with the solid phase samples therein can be moved to a predetermined site and subjected to solid phase procedures selected to analyze or synthesize a nucleic acid. The solid supports of the present invention can be used in parallel and are preferably configured in a 96 cavity or 384 cavity format. The solid supports can be attached to pins, rods or bars in a configuration of 96 cavities or 384 cavities, the solid supports can either be separated or alternatively be integral to the particular configuration. The particular configuration of the solid supports is not of critical importance for the operation of the test, but rather, it affects the ease of adaptation of the tests to automatic systems. 3. Methods for Attaching Nucleic Acid Molecules to a Solid Support The tips described herein are useful in a variety of methods requiring the attachment of a nucleic acid molecule, peptide, polypeptide or protein to a solid support. Examples of solid phase assays and detection methods that can be performed with such tips are described below. Standard methods can be used to attach a nucleic acid molecule to the tips. For example, nucleic acid molecules, modified at their 5 'ends with an aldehyde or carboxylic acid, can be attached to a solid support having hydrazide residues (see, for example, Kremsky et al., Nucleic Acids Res. 15: 2891 , 1987). Alternatively, the 5'-aminophenyl phosphoramidate derivatives of oligonucleotides can be coupled with a solid support carrying carboxyl groups in a carbodiimide mediated coupling reaction (see, for example, Ghosh et al., Nucleic Acids 15: 5353, 1987). ). The solid supports are preferably coated with an amine polymer such as polyethylene (imine), acrylamide, amine dendrimers, etc. The amines in the polymers are used to covalently immobilize nucleic acids. Preferably, the nucleic acids bind to the solid supports described herein using poly (ethyleneimine) (PEI) coatings. The chemistry used to adhere a layer of PEI to the substrate depends, in substantial part, on the chemical identity of the substrate. The prior art provides numerous examples of suitable chemistries that can adhere PEI to a solid support. For example, when the substrate is nylon 6/6, the PEI coating can be applied through the methods described by Van Ness et al., Nucleic Acids Res. 19: 3345, 1991, and international patent application No. WO 94 / 00600. Suitable methods for applying a PEI layer to solid glass or silicon supports are described by, for example, Wasserman, Biotechnology and Bioengineering XXII.271, 1980, and by D'Souza, Biotechnology Letters 8: 643, 1986. Preferably , the PEI coating is covalently bound to the solid substrate. When the solid substrate is glass or silicon, the PEI coating can be covalently bound to the substrate using silylation chemistry. For example, PEI having reactive siloxy end groups is commercially available from Gelest, Inc. (Tullytown, PA). The reactive PEI may be contacted with a glass or silicon tip, and after moderate agitation, PEI will adhere to the substrate. Alternatively, a bifunctional silylation reagent may be employed. According to this process, the glass or silicon substrate is treated with the bifunctional silylation reagent to provide the substrate with a reactive surface. PEI is then contacted with the reactive surface, and covalently attached to the surface through the bifunctional reagent. PEI coatings are preferably used to immobilize nucleic acids on the nylon tips, as described herein. A suitable method for coating nylon 6/6 with PEI has been described by Van Ness et al., Nucleic Acids Res. 19: 3345, 1991. In summary, the nylon substrate is ethylated using triethyloxonium tetrafluoroborate to form imidate esters reactive with amine on the nylon surface. The active nylon then reacts with PEI to form a polymer coating that provides an extended amine surface. After activation of the oligonucleotides with 5'-aminohexyl end with 2,4,6-trichloro-1, 3,5-triazine (cyanuric chloride), the modified oligonucleotides are covalently bound to the nylon surface through the of triazine. Accordingly, the preferred nucleic acid polymers are "amine modified" since they have been modified to contain a primary amine at the 5 'end of the nucleic acid polymer, preferably with one or more methylene groups disposed between the primary amine and the nucleic acid portion of the nucleic acid polymer. Six is the preferred number of methylene groups. The nucleic acid molecules can be modified through the addition of amine moieties using standard techniques. The products of a polymerase chain reaction, for example, can be arranged using initiators modified with 5'-hexylamine. Nucleic acid duplexes can be arranged after the introduction of amines through nick translation using amine-allyl-dUTP (Sigma, St. Louis, MO). The amines can also be introduced into nucleic acids through polymerases such as terminal transferase with amino-allyl-dUTP or through ligation of nucleic acid polymers containing short amine on nucleic acids through ligases. Preferably, the nucleic acid polymer is activated before being contacted with the PEI coating. This can be conveniently achieved by combining the functionalized nucleic acid polymer with amine with a multifunctional amine reactive chemical such as cyanuric chloride. For example, an excess of cyanuric chloride can be added to a solution containing the nucleic acid polymer solution. Preferably, the solution can contain a molar excess of 10 to 1000 times of cyanuric chloride over the number of amines in the nucleic acid polymer in the disposition solution. In this manner, most of the amine terminated nucleic acid polymers have reacted with a molecule of cyanuric chloride, so that the nucleic acid polymer is terminated with dichlorotriazine. An advantageous aspect of the invention is that the disposal solutions containing biomolecules can be deposited on a PEI coating although that arrangement solution contains an important solution of the multifunctional amine reactive chemical. This provides an important advantage over methods where the coupling agent needs to be removed from the disposal solution before a disposal process. When the nucleic acid polymer is of double stranded structure, both structures or one of the chain structures contains a terminal amino group. The nucleic acid polymer of double chain structure can be linked through a terminal amino group to the PEI coating to immobilize the double chain structure polymer. Since only one of the two chain structures is covalently bonded to the PEI coating, the other chain structure can be removed under denaturing and washing conditions. This aspect provides a convenient method according to the present invention to obtain an arrangement of nucleic acid polymers of individual chain structure. The nucleic acid polymer of double chain structure can be obtained, for example, as a reaction product from PCR. Preferably, the arrangement solution is regulated in its pH using a common pH regulator such as sodium phosphate, sodium borate, sodium carbonate, or Tris-HCl. A preferred pH scale for the disposal solution is from 7 to 9, with a preferred pH regulator being recently prepared which is sodium borate at a pH of 8.3 to 8.5. Several methods described below require the use of oligonucleotides attached to the solid supports of the present invention. Preferably, the oligonucleotides are synthesized with a 5'-amine (generally a hexylamine, which is a distant amine and a 6 carbon spacer arm). Typically, the oligonucleotides are from 15 to 50 oligonucleotides in length, and are activated with homobifunctional or heterobifunctional crosslinking reagents, such as cyanuric chloride. The activated oligonucleotides can optionally be purified from an excess of crosslinking reagent (e.g., cyanuric chloride) through exclusion chromatography. The activated oligonucleotides are then mixed with the solid supports to effect covalent attachment. After covalent attachment of the oligonucleotides, the unreacted amines of the solid support are blocked at their end (eg, with succinic anhydride) to remove the positive charge from the solid support. Certain methods require the use of biotinylated oligonucleotides that bind to streptavidin, which, in turn, binds to a solid support. The methods for producing biotinylated nucleic acid and streptavidin molecules bound to the support are well known to those skilled in the art. For example, Van Ness et al., Nucleic Acids Res. 19: 3345 (1991), describe a method for the biotinylation of oligonucleotides, wherein the oligonucleotides are treated with activated biotins. Alternatively, biotinylated oligonucleotides can be prepared by synthesizing oligonucleotides with biotin-labeled dNTPs (see, for example, Ausubel et al., (Eds.), Short Protocols a Molecular Biology, 3rd Edition, pages 12-23 to 12-25 (John Wiley &; Sons, Inc. 1995)). Methods for the biotinylation of nucleic acids are well known in the art and are described in, for example, Avidin-Biotin. Chemistry: A. Handbook (Pierce Chemical Company 1992). Standard methods that can be used to bind streptavidin to the tips of the present invention are provided by, for example, Das and Fox, Am. Rev. Biophys. Bioeng. 8: 165, 1979, and by Wilchek and Bayer. Anal. Biochem. 171: 1, 1983. The tips described herein can also be used to analyze peptides. General guidelines for the conjugation of peptides, polypeptides and proteins to solid supports were tested, for example, by Wong, Chemistry of Protein Conjugation and Cross-Linking (CRC Press, Inc. 1991), and by Partis et al., J. Protein Chemistry 2: 263 (1982). The use of peptides and antibodies in solid phase processes are known from, for example, Vaughn et al., Nature Biotechnology 14: 309, 1996 and House et al., Science 246: 1275, 1989. 4. Use of Solid Supports in the cDNA Synthesis. As described above, there is an increasing need to synthesize cDNA on solid support. These cDNA molecules can be used to create cDNA libraries and as probes for gene expression analysis and diagnostic assays. The design of the solid supports described addresses the following problems in current cDNA-dependent technologies: high input of required RNA, low number of sample production, many user manipulations, extractions and organic precipitations, vector deviation for insert size and poor ability to adapt An advantage of the solid phase aspect is that the synthesis of cDNA in solution requires a large number of steps with intermediate steps of precipitation. There are several aspects for producing a cDNA molecule on a specialized solid support as described in this description. The following general scheme provides an illustration. First, RNA is prepared using standard techniques. In the studies described in Example 1, total RNA was prepared through acid-guanidinium-phenol extraction using well-known methods (see, for example, Ausubel et al. (Eds), Short Protocols in Molecular Biology, 3rd Edition, pp. 4-4 to 4-6 (John Wiley &Sons, Inc. 1995), Wu et al., Methods in Gene Biotechnology, pp. 33-34, (CRC Press 1997)). Then, messenger RNA is captured on a solid support, for example, one containing oligonucleotides having oligo (dT) ends. Alternatively, it is possible to use a simultaneous lysis-mRNA capture protocol using a chaotrope, such as guanidinium thiocyanate or guanidine hydrochloride, both cell lysis and for hybridization. This aspect allows the lysis of a small number of cells. According to this method, DNA was removed by passing the lysate through a fiberglass filter, the mRNA was captured on the solid support, and the unbound contaminants and the material were washed. This avoided losses associated with organic phase extractions and serial ethanol precipitations inherent in standard RNA preparation procedures. The RNA bound to the support was used as a template to produce the first strand structure of the cDNA, using standard methods. Then, the second strand structure of the cDNA was synthesized or an adapter was placed at the distant end of the first strand cDNA. As an illustration it is possible to place three dNGs at the 3 'end of the first chain structure using, for example, terminal transferase. Alternatively, an adapter can be ligated onto the 3 'end of the cDNA molecule. Then, a complementary initiator hybridized to the adapter. The second strand structure of the cDNA was then synthesized using the first strand structure of the cDNA as a template. Alternatively, sequences either random or specific for bound RNA can be amplified using a polymerase chain reaction (PCR). In summary, PCR is a process based on a specialized polymerase, which can synthesize a chain structure complementary to a given DNA strand structure in a mixture containing deoxyribonucleotides and two DNA primers, each with a length of approximately 20 bases, which flank the target sequence. The mixture was heated to separate the chain structures of the double-stranded structure DNA containing the target sequence and then cooled to allow the primers to bind to their complementary sequences on the separate chain structures. The polymerase then extends the primers to new complementary strand structures. Repetitive heating and cooling cycles multiply the target DNA exponentially, since each new double-stranded structure is separated to become two templates for further analysis. In about 1 hour, 20 PCR cells can amplify the target by one million times. Standard methods 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, White (ed.), pp. 1-29 (Humana Press, Inc. 1993); Ausubel et al. (Eds), Short Protocols in Molecular Biology, 3rd. Edition, pages. 15-1 to 15-40 (John Wiley &Sons, Inc. 1995)). Accordingly, an initiator can be added which is complementary to the known part of the bound mRNA. Then, it is possible to amplify using a specific initiator and an initiator complementary to the adapter. After amplification, typically 5-15 rounds of thermocycling, 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 containing sequences that are complementary to the adapter and the 5 'end of the gene. The full-length cDNA is then amplified from the solid support. A modification of PCR, called "anchor PCR", allows the amplification of full length mRNA, although only a small amount of sequence information is available. Ausubel et al. (Eds), Short Protocols in Molecular Biology, 3rd. Edition, pages. 15-27 to 15-32 (John Wiley &Sons, Inc. 1995)). This method requires an oligo (dT) primer that is both complementary to the poly (A) end of the mature mRNA, when amplified downstream to the known sequence, and complementary to one end of the synthesized homopolymer added to the cDNA following first structure synthesis chain, when amplified upstream to the known sequence. There are at least two branch points in these general cDNA synthesis methods, which provide starting linkages for additional techniques that take advantage of the solid support methodology. A branch point follows the synthesis of the first chain structure cDNA. At this point, the remaining RNA template can be digested with RnaseH, hydrolyzed in sodium hydroxide or removed by thermal denaturation, leaving a DNA template of individual chain structure, which can be used for chain structure synthesis secondary oligonucleotide-directed, PCR, randomized initiation probe production, or gene expression studies using labeled oligonucleotides. The bound cDNA can also be used to prepare a subtracted collection or differential probes of various applications. The synthesis of secondary chain structure cDNA represents a second branch point in the solid support cDNA technology. Here, choice can be made to bind adapters to the cDNA that can support processes such as cDNA probes of full-length individual strand structure, collection production, in vitro transcription, and 5'RACE. An important advantage for solid support cDNA synthesis is the ability to automate the process. For high throughput cDNA collection production or gene expression studies, it is useful to adapt the solid support described herein to a 96-well format. A robotic arm can be used to deliver 96 supports on 96 gold-coated pins and direct cDNA synthesis on a standard 96-well Cetus plate.

. Gene Expression Analysis The solid supports described herein can be used in high throughput methods to examine the expression of numerous genes (1-2000) in an individual measurement. These methods can be performed in parallel with more than one hundred samples per process. The method is applicable to drug classification or screening, developmental biology, molecular medicine studies, and the like. Thus, within one aspect of the invention, methods are provided for analyzing the gene expression pattern from a selected biological sample, comprising the steps of (a) exposing nucleic acids from a biological sample, (b) combining the exposed nucleic acids with one or more detectably labeled nucleic acid probes, selected, under conditions and for a time sufficient for the probes to hybridize to the nucleic acids, wherein the detectable label is correlated with a particular nucleic acid probe and detectable through spectrometry or potentiometry, (c) separating the hybridized probes from unhybridized probes, (d) detecting the level of spectrometry or potentiometry, and (e) determining from this the gene expression pattern of the biological sample. Within a particularly preferred embodiment of the invention, tests or methods are provided, which are carried out as follows. RNA from a target source is bound to a solid support through a specific hybridization step (e.g., capture of poly (A) mRNA through a bound oligo capture probe (dT)). The solid support is then washed and the cDNA is synthesized on the solid support using standard methods (i.e., reverse transcriptase). The RNA chain structure is then removed through hydrolysis. The result is the generation of a population of DNA, covalently and mobilized to the solid support, which reflects the diversity, abundance and complexity of the RNA from which the cDNA was synthesized. The solid support was then hybridized with one to several hundred probes which are complementary to a gene sequence of interest. Each type of probe was labeled with a detectable label through the spectrometric method, such as mass spectrometry. After the interrogation step, the excess or non-hybridized probe was washed, the solid support was placed, for example, in the cavity of a microtiter plate and the detectable label was separated from the solid support. The solid support was then removed from the cavity of the sample container, and the contents of the cavity were measured with a spectrometer. The appearance of specific labels indicates the presence of RNA in the sample and evidence that a specific gene is expressed in a given biological sample. The method can also be quantifiable. The compositions and methods for rapid measurement of gene expression using cleavage capable labels can be described in detail as follows. In summary, tissue, primary or transformed cell lines, types of isolated or purified cell or any other source of biological material where the determination of gene expression is useful, can be used as a source of RNA. In the preferred method, the biological source material is lysed in the presence of a chaotrope to suppress nucleases and proteases, and to support severe hybridization of the target nucleic acid to the solid support. The tissues, cells and biological sources can be effectively used in 1 to 6 molar chaotropic salts (guanidine hydrochloride, guanidine thiocyanate, sodium perchlorate, etc.). After the source biological sample is lysed, the solution is mixed for 15 minutes to several hours with the solid support to effect the mobilization of the target nucleic acid. In general, the capture of the target nucleic acid is achieved through complementary pass pairs of Target RNA and the capture probe immobilized on the solid support. A permutation utilizes the 3'-poly (A) stretch found in most eukaryotic messenger RNAs to hybridize to an oligo (dT) bound on the solid support. Another permutation is to use a specific oligonucleotide or long probes (greater than 50 bases) to capture an RNA containing a defined sequence. Another possibility is to use degeneration primers (oligonucleotide) that could capture numerous related sequences in the target RNA population. For example, the RNA samples can be transcribed in reverse with each of the four groups of oligo (dT) initiators anchored in degeneration, having the formula 5'-T12MN-3 ', where M can be G, A or C, and N is G, A, T and C (Ausubel et al. (eds), Short Protocols in Molecular Biology, 3rd Edition, pp. 15-35 to 15-40 (John Wiley &; Sons, Inc. 1995)). Each group of initiators dictated by base 3 ', with the degeneracy in the M position. Hybridization times are guided by the complexity of the sequence of the RNA population and the type of capture probe employed. Hybridization temperatures are dictated by the type of chaotrope used and the final concentration of the chaotrope. General guidelines are provided, for example, by Van Ness and Chen, Nucleic Acids Res. 19: 5143, 1991. The lysate is preferentially agitated with the solid support continuously to effect diffusion of the target RNA. After capturing the target nucleic acid, the lysate is washed from the solid support and the entire chaotrope or hybridization solution is removed. The solution support is preferentially washed with solutions containing ionic or non-ionic buffering agents and salts. The next step is the synthesis of DNA complementary to the captured RNA, wherein the bound capture oligonucleotide serves as the extension primer for reverse transcriptase. The reaction is generally carried out at 25 to 37 ° C, and preferably it is stirred during the polymerization reaction. After the cDNA is synthesized, it is covalently bound to the solid support, since the capture oligonucleotide serves as the extension primer. Then, the RNA is hydrolyzed from the duplex cDNA / RNA. The step can be effected through the use of heat, which denatures the duplex or the use of base (i.e., 0.1 N NaOH) to chemically hydrolyze the RNA. The goal of this step is to make the cDNA available for subsequent hybridization with defined probes. The solid support or solid support group is then further washed to remove the RNA or RNA fragments. In this point. The solid support contains an approximate representative population of cDNA molecules representing the RNA population in terms of sequence abundance, complexity and diversity. The next step is to hybridize selected probes to the solid support to identify the presence or absence and the specific relative abundance of cDNA sequences. The probes are preferably oligonucleotides with a length of 15 to 30 nucleotides. The sequence of the probes is dictated by the end user of the test. For example, if the end user intends to study gene expression in an inflammatory response in a tissue, the probes can be selected to be complementary to numerous cytokine mRNAs, ARNS encoding lipid modulating enzymes, RNAs encoding factors that regulate cells in an inflammatory response, etc. Once a group of desired sequences is defined for study, each sequence is used to design an oligonucleotide probe, and each probe is assigned a label capable of specific cleavage. The tag (s) is then linked to the respective oligonucleotide (s). The oligonucleotides are then hybridized to the cDNA on the solid support under appropriate hybridization conditions. After completing the hybridization step, the solid support is washed to remove any unhybridized probe. The solid support or support arrangement is then placed in solutions that effect the cleavage of the detectable labels. The presence (and abundances) of an expressed mRNA is determined by measuring the amount of detectable labels. For example, mass spectrometer labels are examined using a mass spectrometer. There are numerous variations of the method described above for analyzing differential expression. For example, the differential expression can be examined using a subtracted collection. Subtraction of redundant messages to reveal gene expression patterns representative of particular activation or development states is a desirable capability of any gene discovery program. There are many protocols for the production of subtracted ADNx collections, but many require either large amounts of RNA or pre-existing cDNA collections. The use of a solid support to capture mRNA allows the message source representing the antecedent to be subtracted. The desired mRNA species are reverse transcribed and the RNA templates are destroyed with alkali. The resulting "subtraction template" can be reused indefinitely. To make a subtracted collection, the RNA from the source under investigation is denatured with heat, then hybridized to the first strand structure of the cDNA in the subtraction template. The unbound RNA can then be washed and either directly captured or hybridized a second time after all the subtracted or bound RNA has been eluted from the subtraction template. After capture the synthesis of the cDNA continues as previously described. In the related aspect, the subtraction cDNA probes are prepared by hybridizing the individual chain structure cDNA from a cell type with immobilized mRNA from a closely related cell type, and isolating the small fraction from the non-cDNA. hybridized As a result of this enrichment, subtracted cDNA DNA fragments can be used to identify cDNA clones containing differentially expressed sequences. Subtraction cDNA can also be used to prepare collections of subtraction cDNAs. PCR can be used to amplify the subtraction cDNA for use as probes or for cloning (see, for example, Kuel and Battey, "Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA", a PCR Protocols: Current Methods and Applications , White (ed), pp. 287-304 (Humana Press, Inc. 1993); Wu et al., Methods in Gene Biotechnology, pgs. 29-65, (CRC Press 1977)). In another variation, a biotinylated oligo (dT) MN molecule, the degeneracy initiator described above, is used to bind mRNA to a solid support, and to initiate the synthesis of first chain structure (see, for example Rosok et al., BioTechniques 27: 114, 1996). After reverse transcription, the solid phase cDNA is used as a template in a polymerase chain reaction, with the biotinylated oligo (dT) MN and an arbitrary decamer as primers. The PCR products obtained from two cell populations were then compared following fractionation through polyacrylamide gel electrophoresis, the PCR bands of interest were eluted from the gel, and purified PCR products were used as templates for a chain reaction of additional polymerase, which amplifies the selected bands. The products of the second polymerase chain reaction can be used as probes, or can be further examined through sequence analysis. 6. Solid Phase Diagnostic Analysis. (A) Detection of Polymorphisms Restriction endonucleases recognize short DNA sequences and separate DNA sequences at those specific sites. Certain restriction enzymes stop the DNA in a very unusual way, generating a small number of very large fragments (several thousand to one million base pairs). Most restriction enzymes separate DNA more frequently, thus generating a large number of small fragments (less than 100 to more than 1000 base pairs) on average, restriction enzymes with four base recognition sites will produce pieces with a length of 256 bases, six base recognition sites will produce pieces of length of 4000 bases, and eight base recognition sites will produce pieces with a length of 64,000 bases. Since hundreds of different restriction enzymes have been characterized, DNA can be separated into many different small fragments. Although some known human DNA polymorphisms are based on insertions, deletions or other rearrangements of non-repeated sequences, the vast majority is based on either individual base substitutions or variations in the number of tandem repeats. Base substitutions are very abundant in the human genome, occurring on average once every 200-500 base pairs. Length variations in blocks of tandem repeats are also common in the genome, with at least 10,000 interdispersed polymorphic sites, or "places." The repetition lengths for tandem repeat polymorphisms vary from a base pair in sequences (dA) n (dT) n to at least 170 base pairs in the alpha-satellite DNA. Tandem repeat polymorphisms can be divided into two main groups, which consist of mini-satellites / variable number of tandem repeats (VNTRs), with typical repetition lengths of ten base pairs and with 10,000 repeat units, and microsatellites, with repetition lengths of up to 6 base pairs and with maximum total lengths of approximately 70 base pairs. Most of the microsatellite polymorphisms identified to date have been used in dinucleotide repeat sequences (dC-dA) n or (dG-dT) n. The analysis of microsatellite polymorphisms involves the amplification through the polymerase chain reaction of a small fragment of DNA containing a block of repeats followed by electrophoresis of the DNA amplified in denatured polyacrylamide gel. PCR primers are complementary to unique sequences flanking the repeating blocks. Polyacrylamide gels, instead of agarose gels, are traditionally used for microsatellites, since alleles usually differ in size only by individual repetition. A wide variety of techniques has been developed for the analysis of DNA polymorphisms. The most widely used method, the appearance of. Restriction fragment length polymorphism (RFLP), combines restriction enzyme digestion, gel electrophoresis, membrane staining to a membrane and hybridization to a cloned DNA probe. Polymorphisms are detected with variations in the lengths of the fragments marked in the stains. The RFLP aspect can be used to analyze base substitutions when the sequence change falls within a restriction enzyme site, or to analyze minisatellites / VNTRs by selecting restriction enzymes that cut the repeat units. Agarose gels usually do not offer the resolution necessary to distinguish minisatélite / VNTR alleles differing by an individual repeating unit but many of the minisatellites / VNTRs are thus variables that highly informative markers can still be obtained. (You and others, Nuc Acids Res. 23: 4407, nineteen ninety five). Solid phase techniques have improved the ability to detect polymorphisms. For example, a biotinylated primer with specific allele PCR primers is used to amplify a form of an allele. After amplification, the PCR products can be detected through hybridization of solution to a fluorophore labeled probe, and the hybrids are captured on a solid phase support carrying streptavidin (see for example, Syvanen and Landegren, Human Mutation 3 : 172, 1994). In another aspect, "solid phase minisequencing", a biotinylated amplification product is immobilized by a support coated with streptavidin. The amplification product is then used as a template in a specific sequence extension reaction in the presence of an individual nucleoside triphosphate implemented with one of the sequence variants that will be distinguished (see for example, Syvanen and Landegren, Human Mutation 3: 172, 1994; Syvanen, Clin. Chem. Acta 226: 225, 1994; Jarvela and others J. Med. Genet. 33: 1041, 1996). In the "oligonucleotide ligation assay", two different differentially labeled specific allele oligonucleotides are compared for their ability to bind to a biotinylated downstream oligonucleotide (see, for example, Nickerson et al., Proc. Nati. Acad. Sci. USA 87 : 8923, 1990, Nickerson et al., Genomics 12: 377, 1992). The unlabeled oligonucleotide is immobilized on a solid support coated with avidin which projects into a test cavity. The presence of the target sequence is determined by measuring the particular signal generated by the bound labeled oligonucleotide. For example, specific allele oligonucleotides can be labeled with different fluorophores, and the presence of the target is determined by measuring the fluorescence. Nepom et al., J. Rhematol. 23: 5 (1996), describe a method for genotyping assays in which a selected sequence is amplified using PCR with a biological sample of nucleic acid and a biotinylated primer. A small amount of amplified product is transferred to an automatic precursor instrument that performs the reduction and detection of specific allele.

The fingerprint imprint of DNA represents another aspect of polymorphism detection. A variety of DNA fingerprint printing techniques are now available, many of which use PCR to generate fragments (see for example, Jeffries et al., Nature 314: 67, 1985; Welsh and McClelland, Nucleic Acids Res. : 861, 1991). The choice of such a finger print technique to be used depends on the application (eg, DNA typing, DNA marker mapping), and the organisms under investigation (eg, prokaryotes, plants, animals, humans). In general, DNA fingerprinting can be performed by synthesizing full-length cDNAs on a tip of the present invention. The cDNA is then digested with restriction endonucleases, and the unbound material is rinsed from the tip. Then, adapters are ligated onto the bound cDNA, and the product can be amplified and analyzed. A number of fingerprint printing methods have been developed in recent years, including random amplified polymorphic DNA (RAPD), DNA amplification finger print (DAF), and arbitrarily initiated PCR (AP-PCR). These methods are based on the amplification of random genomic DNA fragments through arbitrarily selected PCR primers. DNA fragment patterns can be generated from any DNA without prior knowledge of sequence. The generated patterns depend on the sequence of the PCR primers and the nature of the template DNA. PCR is performed at low heating and cooling temperatures to allow the primers to warm and cool in multiple places on the DNA. The DNA fragments are generated when the primer binding sites so within a distance allow amplification. In principle, a single initiator is sufficient to generate band patterns. A more recent technique for printing DNA fingerprints and for detecting polymorphism is the amplified fragment length polymorphism (AFLP) technique "see for example, Vos et al., Nucleic Acids Res. 23: 4407, 1995; Schreiner and others, J. Immunol, Methods 196: 93, 1996.) In summary, genomic DNA is digested with restriction endonuclease and ligated with oligonucleotide adapters, PCR provides the selected amplification of groups of restriction fragments and amplified fragments are analyzed after fractionation through polyaclamide gel electrophoresis This method is easily adapted to a solid phase using the tips of the present invention Here, the full-length genomic DNA is covalently immobilized on a tip. then it is digested with restriction endonucleases, and the unbound material is washed from the tip, then the adapters are bound to the bound genomic DNA fragments, and the bound DNA molecules are amplified and analyzed. The AFLP technique has also been applied to the imprinting of mRNA trace (Habu et al., Biochem. Biophys. Res. Commun. 234: 516, 1997). In this regard, the double-stranded structure cDNA was synthesized with anchored oligo (dT) primers, and digested with Taql, which recognizes a four-base sequence. A Taql adapter is then ligated to the ends of the cDNA fragments, and amplification is performed by PCR with selected primers, following the general methods of AFLP-based genomic fingerprint printing. Advantageously, the imprinting of mRNA trace through the AFLP technique was performed with the solid supports described herein to anchor the oligo (dT) primers. Mutations can also be identified through their destabilizing effects on the hybridization of short oligonucleotide probes to an objective sequence (see, for example, Wetmur, Crit., Rev. Biochem.Mol. Biol. 26: 227, 1991. In general , this specific allele oligonucleotide hybridization technique involves the amplification of target sequences and subsequent hybridization with short oligonucleotide probes.An amplified product can be screened for many possible sequence variants by determining its hybridization pattern to an oligonucleotide probe array immobilized The illustrations of this aspect are given in Examples 6 and 7.

(B) General Diagnostic Methods. DNA probes can be used to detect the presence of infectious agents or diseased cells, such as tumor cells expressing antigens associated with the tumor. Typically, a biological test sample is subjected to a lysis step using ionic detergents or coatropes to release the nucleic acid targets. Typical nucleic acid targets include mRNA, genomic DNA, plasmid DNA or RNA, and viral rRNA, DNA or RNA. To perform the detection of the target nucleic acid, the target requires some type of immobilization. For example, nucleic acids are immobilized on a solid support or substrate that possesses some affinity for the nucleic acid. The solid supports are then probed with labeled oligonucleotides of predetermined sequence to identify the target nucleic acid of interest. The non-hybridized probe is removed in a washing step, the labels are separated from their respective probes, and then measured (for a review see, Reischi and Kochanowski Molec, Bíotech, 3:55, 1995). In a general type of assay method, oligonucleotides that represent a characteristic part of an amplified sequence are bound to a solid support. The binding can be covalent or through biotin: streptavidin, or a similar type of binding. The target nucleic acid is used as a template to produce detectably labeled PCR products. These PCR products are hybridized with the capture oligonucleotides, and the presence of the PCR products is determined by a mediated detection reaction.

In a variation of this aspect, the biotin-labeled PCR products are attached to a solid support coated with streptavidin. The immobilized PCR products are hybridized with a labeled probe complementary to internal sequences of the amplification product. As an illustration of a diagnostic method, Wilber, Immunol. Invest. 25: 9 (1997), discloses a solid-phase nucleic acid hybridization assay based on branched DNA signal amplification methods. In this study, HIV RNA was detected in the plasma through hybridization of multiple oligonucleotides to the target, 10 of which captured the target on the surface, and 39 of which mediated the hybridization of the branched DNA molecules to the region. RNA pol. The detectably labeled probes were attached to each arm of the branched DNA molecules. Those skilled in the art are well aware of additional detection methods. For example, solid phase detection can be achieved using amplified colorimetric means, such as an alkaline phosphatase system, a streptavidin system, a radish peroxide system. Radiometric detection is another alternative. Radiolabels suitable for radiometric detection include 3 H, 125 I, 131 I, 35 S, 14 C, 35 P, and the like. Molecules labeled with fluorescein provide other means of detection. The present invention, generally so described, will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES EXAMPLE 1 Synthesis of cDNA On Solid Support (A) RNA capture In these studies, total RNA was prepared through acid-guanidinium-phenol extraction, using standard techniques (see for example, Ausubel et al. (Eds), Short Protocols in Molecular Biology, 3rd Edition, pp. 4-4 to 4-6 (John Wiley &Sons, Inc. 1995)). The polyadenylated mRNA was captured on the tips by first heating the mixture of the isolated total RNA at 70 ° C, adding a solution of hybridization with a high salt content to the RNA, adding the RNA to the solid support having oligo (dT), and then placing the support on a moving platform, such as a rotating mixer. A sufficient mixer was achieved through a variety of instruments including a continuous swirl former, orbital agitator, rotary oscillator and a hybridization oven equipped with a rotating apparatus. The time required for RNA capture was found to depend on the amount of input RNA. At room temperature, 10 μg of the total RNA was sufficient to achieve a 90% saturation of a tip in about 2 hours. In a typical test cell, this amount of total RNA corresponds to approximately 100 ng of poly (A) plus mRNA attached per tip. Forty micrograms of total RNA from the same source gave the same level of saturation in 30 minutes. In another series of experiments, a stimulated human T cell line was used as the RNA source and saturation was achieved in one hour under the same capture conditions using an entry of 10 μg of RNA. Several additional RNA sources were captured including mouse, hamster and human cell lines, all tend to fall within the 90% capture range in 1-2 hours per 10 μg of total RNA, defining a time of maximum incubation and minimum one.

(B) DNA Synthesis of First Chain Structure After capture of poly (A) plus mRNA, the tip was washed in pH buffer hybridization 3 times to remove unbound RNA. Reverse transcription was performed in a volume of 30 μl containing MMLV-reverse transcriptase (MMLV-RT) and an optimized pH-regulator system of 1-2 hours at 42 ° C on a rotating hybridization furnace support. As with the capture step, for a poor synthesis of the first chain structure requires a constant mixing of the reagents. The initial experiments produced 50-100 ng of cDNA per solid support. The products of the reverse transcription were indirectly analyzed through autoradiography of the cDNA of double-labeled structure separated from the tip and sized through agarose gel electrophoresis. In several experiments, the extended size of the copied mRNA species was comparable to conventional methods and was generally larger. The cDNA size distribution varied from 0.5 kilo bases-20 kilo bases, with an average being approximately 2.0 kilo bases.

(C) Synthesis of Second Chain Structure cDNA After reverse transcription, the tip was washed three times to remove the reagents and the enzyme. The second chain structure synthesis was performed in a volume of 40 μl using one unit of RNaseH per 25 units of polymerase I of E. coli DNA. The reaction was incubated at room temperature on a rotary oscillator for 6 hours or overnight. The non-uniformly extended ends were "polished" for the subsequent ligation step, removing the reaction of the second chain structure and adding T4 DNA polymerase and dNTPs. This incubation was continued for 30 minutes at 37 ° C on the rotary apparatus in a hybridization oven. The products were visualized directly by running a reaction in the presence of 32 P-labeled dNTP, and either by boiling the products of secondary chain structure, or by cleaving the appropriate support with the restriction enzyme Ascl. The radiolabeled products were run on a gel and placed on a film for visualization. From a typical input of 10 μg of total RNA, it is possible to recover 50-120 ng of double-stranded structure cDNA, depending on the particular RNA. The use of a thermostable DNA polymerase after reverse transcription with an enzyme having an RNaseH activity, such as MMLV-RT is also a possibility. In this case, the thermostable polymerase digests the temperature of the RNA, eliminating the need to remove the RNA through a step of thermal denaturation. An experiment comparing RNaseH / DNA, pol 1 against Tthl DNA polymerase in a second synthesis of chain structure, showing little difference both in the quality of the product divided by Ascl and the content of that product, which was tested through amplification PCR of selected genes such as GAPDH and IL-2. Using Tthl reduces the incubation time from 6 hours at room temperature to 1 hour at 70 ° C. Tthl polymerase also shows reverse transcriptase activity in the presence of manganese ions, but incubations need to be maintained for very little due to the possibility of high temperature RNA hydrolysis in the presence of divalent cations. Another advantage for carrying out DNA polymerization at high temperature is that the secondary structure is reduced, which helps the synthesis of highly structured messages.

(D) Ligation of Vector Adapter-Ligation to the Vector Hemi-phosphorylated adapters were ligated to the double chain structure cDNA in volume of 30 μl with a molar ratio of adapted cDNA of 5.10: 1. A preferred ligation pH regulator contained 10% PEG. The adapter-cDNA mixture was incubated with T4 DNA ligase overnight at room temperature on a rotary oscillator. The solid support was then washed three times to remove excess linkers. After the ligation, the 5'-hydroxyl group in the adapter was phosphorylated with T4 polynucleotide kinase and ATP for 1 hour at 37 ° C on a hybridization furnace rotation support. This reaction was stopped by washing the tip three times in pH buffer of TE. If the cDNA is to be used for another application, the phosphorylation step may not be necessary. In some applications, the synthesis of secondary chain structure was specifically initiated from an adapter added immediately after reverse transcription. According to this aspect, after hydrolysis of the mRNA, a heteroduplex adapter, of partially individual chain structure, can be ligated using T4 RNA ligase. The nature of the partial double-stranded structure of this adapter can provide an idroxy 3'-h group I or from which the synthesis of the second strand structure can begin, and could prevent concatemerization of the adapter during ligation, since the T4 RNA ligase can not bind nucleic acids of double-stranded structure.

(E) Support cleavage / recirculation In certain studies, a vector was ligated to the cDNA that had been synthesized on a tip. The cDNA or vector: cDNA was separated from the solid support in a volume of 40 μl with Ascl at 37 ° C on a rotary rack of hybridization oven for 4 hours. The separated DNA was then heated at 70 ° C for 20 minutes with or without mixing. The vector: cDNA was recirculated directly bringing the volume to 50 μl with the addition of ligation pH regulator and T4 DNA ligase. The cDNA previously not bound to the vector was divided into several aliquots for test ligations to determine which vector: insert relationship gives the best result in subsequent transformations. In this case, since the total amount of cDNA is small (50-120 ng total in volume of 4 μl), the entire ligation must be transformed, making some kind of desalination step necessary. It is possible to separate more than 90% of the cDNA from a tip in 4 hours. This seems to be not advantageous for increasing the incubation time under these conditions. Increasing the enzyme concentration can cut the time necessary for cleavage, but an equilibrium should be pursued between the incubation time and the volume of excision.

(F) Transformation The cDNA clones were propagated through electroporation transformation of the electrocomponent, E. coli, with aliquots of DNA from a ligation. The transformation frequencies can vary between 109-101 ° cfu per μg of DNA. As those skilled in the art know, the main limitation in this procedure is the salt sensitivity of electroporation. Only 1-2 μl of a standard ligation (5-10% of total volume) can be electrophoresed by electrocompetent cells before the threshold salt tolerance and current arcs applied between the electrodes are exceeded, vaporizing the cells and washing the ligation portion used. Many separate aliquots of cells can be electroformed so that this does not happen, but this is both laborious and exhausting. A better scheme is to use a desalination step that can be added to the current cDNA methodology, which is flexible enough to be adapted to an online version or escalation of the technology without sacrificing production. The transformants were plated onto the standard growth medium, such as antibiotics containing LB agar. Only bacteria that host the gene for antibiotic resistance form colonies in media containing that antibiotic. The differentiation between true recombinants and colonies containing only vector sequences, is usually achieved through the selection of blue-white color, which is a result of the expression of the lacZ gene, which runs through the sites of multiple cloning of many common plasmid vectors. The expression of the lacZ gene product is interrupted by an insert linked to the multiple cloning site resulting in a white colony phenotype. In this scenario, the recombinants can be visually identified from non-recombinants, but they must be collected from the non-recombinants or a certain level of prior non-recombinants must be tolerated in the final product. Still, a relatively low background of non-recombinants can cause problems when the collection is amplified, since vectors containing bacteria without inserts tend to be faster in their development and are more stable than those containing inserts, especially those with recombinants inserts. big.

EXAMPLE 2 Climbing Input RNA for cDNA Synthesis on Solid Support By minimizing the loss of material during the many manipulations involved in the production of standard cDNA collection, it is possible to scale the input RNA levels from 10 to 100 times. For example, absolutely 100 g of the double-stranded structure cDNA can be synthesized on a solid support from 10 μg of total RNA, which is 50 times less the starting material than that denominated for commercially available equipment. It may be possible to scale this as much as 10 times without changing the protocol described above significantly, although this will probably require the addition of an amplification step. An alternative method is to follow cDNA synthesis through an adapter ligation using a T7-adapter promoter. This could allow in vitro amplification by transformation in place by PCR, which can produce a more representative collection by eliminating the inherent deviation length in the PCR amplifications. Transcripts synthesized in vitro can then be captured and processed just like another source of RNA. The success of this amplification can be based on the careful optimization of in vitro transcription conditions to ensure full-length transcripts. The non-captured transcripts can be polyadenylated using yeast pilo polymerase (A), then captured by adding them back to the same support. Although newly adenylated transcripts may not be full length, not all of their information can be lost from the RNA combination. In one study, a T7-adapter promoter was synthesized and ligated to double-stranded structure cDNA from 10 μg of total RNA on a tip. By using the conditions suggested by the manufacturer for in vitro transcription, a significant amount of transcripts was produced that ranged in size from 300 base pairs to 2000 base pairs. Although these sizes are not optimal for an effective amplification step, the study shows that the production of in vitro transcripts is possible on a solid support. Another strategy for scaling is to take advantage of asymmetric (linear) PCR in the same way that in vitro transcripts amplify and recapture the product. Since the amplified product is DNA instead of RNA, a DNA polymerase can be used to generate double-stranded structure cDNAs.

EXAMPLE 3 Solid Support Probes The poly (A) plus mRNA of 10 μg of total RNA was captured, reverse transcribed, the secondary chain structure DNA was synthesized, and a T7-adapter promoter was ligated to the cDNA.

In one study, the solid support was placed in a PCR tube of Cetus standard, (ABl, Foster City, CA) and cycled for 35 laps using a large PCR polymerase (EXTaq.TAKARA) in a format, where the extension steps at 70 ° C were increased in length 1 minute per each 5 cycles. Only one primer used in the PCR was complementary to the adapter, so that the amplification can be started from the end 'of the joined cDNAs, producing many copies of the chain structure (+). After cyclization, the PCR products were denatured with heat and run on an agarose gel to visualize the length scale of the individual chain structure cDNA. Sizes ranged from approximately 500 base pairs to more than 20 base kilo pairs, which was in accordance with the sizes of double-stranded structure cDNAs labeled, divided, electrophoresed and visualized through autoradiography in control experiments parallel. To determine whether the PCR product of individual strand structure was representative or not of the original mRNA population, the PCR primers were designed for several genes that are known to be present at a high level, low level, not at all. The design of the primers was such that all products had an approximately equal length (400-600 base pairs) and may be suitable at or near the 5 'end of the cDNA. This initiator design provided a good approximation of the quality of the synthesis of the first chain structure. Since the source of RNA was the human Jurkat cell line, T of IL-2, IL-4, GM-CSF, GAPDH, CTLA4, c-fos, and Werner's helicase sequences were used for the primers. Mouse guanylate kinase was used as a negative control. All polymerase chain reactions produced the expected product size with the exception of CTLA4 and mouse guanylate kinase, as expected. The product for IL-2 was confirmed through Northen staining, where 50 ng of the putative IL-2 was 32P-labeled and hybridized to a stain containing the immobilized RNA from both Jurkat (PMA + ionomycin) and unstimulated Jurkat . After a highly severe wash, the unstimulated RNA showed almost no signal, whereas the stimulated sample presented an intense signal consistent with the expected size for the IL-2 message.

EXAMPLE 4 Use of Solid Support for Rapid Amplification of the cDNA Ends Rapid amplification of cDNA ends, or RACE, is also a technique that can be adapted to the solid support cDNA technology described herein. Both the 5 'or 3' RACE can be performed after ligation of the double stranded structure cDNA adapter using the trailing oligonucleotide rear end (3'-RACE) or the appropriate 5 'adapter oligonucleotide (5'-). RACE) for anchors. The RACE of solid support offers advantages over the techniques currently available, since little material is used to generate the cDNA and the product can be reused. This application can be based on the ability to run a PCR directly on the solid support described above. Initial experiments have shown that a high copy number gene, GAPDH, can be made, and that the final product is directly proportional to the amount of RNA captured and the cDNA is subsequently synthesized. The standard methods for performing 3'-RACE and 5'-RACE are well known to those skilled in the art (see, for example, Wu et al., Methods a Gene Biotechnology, pp. 15-28, (CRC Press 1997)) . EXAMPLE 5 Solid Support cDNA synthesis for the Gene Expression Assay (A) Cell Stimulation and RNA Preparation One group of studies, Jurkat whose line is JRT 3.5, was stimulated for 6 hours at a cell density of 1 x 10 at 6 cells / ml in serum-free RPMI medium ( Life Technologies, Gaithersburg, MD) in the presence of 10 ng / ml of phorbol-12-myristate-13-acetate (Calbiochem, San diego, CA) and 100 ng / ml of ionomycin (Calbiochem). The cells were pelleted, washed in 1 x PBS (Life Technologies), re-formed into pellets and stained in 0.5 ml × 10 6 cells with pH buffer containing 4 M guanidine isothiocyanate / 1% N- lauryl sarcosine / 25 mM sodium citrate (pH 7.1) (Fisher Scientific, Pittsburg, PA). One-tenth volume of 2M sodium acetate (pH 4.2) (Fisher Scientific) was added followed by one volume of phenol saturated with water (Amresco, Solon, OH). After mixing, a quarter volume of chloroform: isoamyl alcohol (29: 1) was added (Fisher Scientific), the solution was mixed vigorously, then incubated on ice for 10 minutes. The lysate was then centrifuged, the aqueous phase was stirred and extracted with an equal volume of chloroform: isoamyl alcohol. The aqueous phase was then poured and the RNA was precipitated with two volumes of ethanol (Quantum Chemical Corp. Tuscola, IL). After centrifugation, the ethanol was decanted and the RNA was air-dried briefly, then resuspended in water-free RNase at a concentration of between 1 and 5 mg / ml.

(B) Capture and Synthesis of First Chain Structure A solid support carrying the oligonucleotide covalently linked, 5? CTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTTT-3 '[SEQ ID NO: 1] (Genset, La Joya, CA), was added to 10 μg of cellular RNA total, and diluted in enough water-free RNase to cover the tip in a sterile 1.5 ml microfuge tube (Fisher Scientific). This oligonucleotide contained a separate and a 5 'Ascl cleavage site from the Oligo (dT) sequence. RNA and tip were incubated at 65 ° C for 5 minutes. An equal volume of 2x pH regulator hybridization mRNA consisted of 50 mM Tris (pH 7.5), 1 M NaCl (Fisher Scientific) of ethylated BSA (New England Biolabs, Beverly, MA) was added to each tube, and tubes were rocked moderately for 2 hours at room temperature. The supernatant was removed and the tip was then washed 3 times a 1x hybridization buffer mRNA. After completing the final wash, a reverse transcription mixture consisting of 1x MMLV-reverse transcriptase pH regulator, 1 mM of a mixture of dNTP, 2 mM of DTT (Life Technologies), 20 units of (Promega, Madison, Wl ) and 10 μg / ml acetylated BSA (New England Biolabs) were added to each tube followed by the addition of 600 units of MMLV-reverse transcriptase (Life Technologies). This reaction was rocked moderately at 42 ° C for 2 hours. A unit of RNase H (Boehringer-Mannheim, Indianapolis, IN) was then added and the reaction was allowed to continue for another half hour. The supernatant was removed again and each tip was washed three times in 10 mM Tris (pH 8) with 1 mM EDTA (pH () (Fisher Scientific) The remaining RNA template was removed by boiling the tips in pH buffer of TE with 0.01% SDS (Fisher Scientific).

EXAMPLE 6 Low Performance High Fusion Procedure Using Solid Support A capture oligonucleotide (36-mer) was covalently linked to a nylon pin assembly coated with polyethylene imine through a C6-amine end. The shorter oligonucleotides (18-mer) were labeled through a C6-amine end with Texas red and were hybridized to the capture oligonucleotide in a 1.5 M solution of guanidinium thiocyanate for 15 minutes at room temperature. The pin assemblies were then washed to remove the unhybridized signal oligonucleotide twice with TEN pH buffer (0.01 M Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl) and then once with pH regulator TENS ( 0.01 M Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl, 0.1% SDS) followed by two washes with TEN pH regulator. The test solutions were aliquoted into cavities of a polycarbonate thermocavities plate (Corning Costar Corp. Cambridge, MA), and the plate was placed in a MJ thermal cycler (MJ Research Company Watertown, MA). The pins were serially transferred between the plate cavities. Every 5 minutes, the temperature increased by 5 ° C, starting at 10 ° C and reaching 85 ° C at the end point. The liquid was transferred to a black 96-well microtiter plate and the fluorescence was measured. The level of fluorescence in each cavity correlates with the amount of signal oligonucleotide that has been fused from the capture oligonucleotide. The "fusion" or duplex dissociation was conducted on a temperature scale of 10 ° C to 95 ° C. Fluorescence was measured with a commercial fluorescence plate reader. To calculate Td, cumulative accounts eluted at each temperature were plotted against temperature. The temperature at which 50% of the material was dissociated from the tip was taken as Td. The helical bovine transition is defined as the temperature at which an alpha value is equal to 0.2 for a given oligonucleotide duplex (or nucleic acid duplex containing or not containing a mismatch anywhere in the duplex) at the temperature a which a value for alpha is equal to 0.8 for the same given oligonucleotide duplex (or nucleic acid duplex). The data was exported to a spreadsheet and fusion curves were generated for each solution. From these melting curves, Td,? HCT, and? Td In a study using a 1 x 12 pin assembly, each test solution was placed in 16 separate cavities of 2 thermocavity plates (Corning Costar Cambridge, MA), containing 100 μl aliquots, one tube for each temperature point . The pin assembly was transferred to a new row of the plate before each temperature jump. Just before reaching a temperature point of 50 ° C, the first plate was removed from the thermal cycler and replaced with the second thermal cavity plate. When the thermal cyclization program was complete, the liquid was transferred to cavities of two black microfluor plates (Dynatech). Fluorescence was measured using an excitation wavelength of 584 nm and an emission wavelength of 612 nm. The data was exported to a spreadsheet program for analysis. In a study using a 4 x 12 pin assembly, 8 thermo cavity plates were cut in half to make 16 4 x 12 cavity plates. An aliquot of 100 μl for each test solution was placed in a cavity of each half of plate until all the cavities contained the test solution. The 16 plate halves were all identical in solution configuration. The pin assembly was transferred to a new half plate before each temperature jump. When the thermal cyclization program was completed, the liquid was transferred to the cavities of 8 black microtiter plates (Dynatech). The fluorescence was measured using an excitation wavelength of 584 nm and an emission wavelength of 612 nm. The data was exported to a spreadsheet program for analysis.

EXAMPLE 7 Determination of the melting temperature of the oligonucleotide duplex in various hybridization solutions based on hibotropic and without hibotrope This example describes the determination of the Td of wild type and mutant nucleotides when hybridized to a target nucleic acid. It was shown that the hybridization solutions based on hibotrope allow the detection of mutations of individual base pairs in a nucleic acid target with a probe up to 30 nucleotides in length.

(A) Solutions and Reagents The filter wash (FW) was 0.09 M NaCl, 540 mM Tris (pH 7.6), 25 mM EDTA. "SDS / FW" is the filter wash with 0.1% sodium dodecylsulfate (SDS). Hybridization solutions contained the concentration specified in the text of hibotrope, 2% N-lauryl sarcosine (sarcosil), 50 mM Tris (pH 7.6 and 25 mM EDTA.

The formamide hybridization solution contained 30% formamide, 0.9 M NaCl, 40 mM Tris-HCl (pH 7.6), and mM EDTA, and 0.1% SDS. Guanidinium thiocyanate was purchased from Kodak (Rochester, NY). GuCI, lithium hydroxide, trichloroacetic acid, NaSCN, NaCIO and Kl, were purchased from Sigma (St. Louis, MO). Rubidium hydroxide was purchased from CSF Chemicals (Columbus, OH). CsTFA was purchased from Pharmacia (Piscataway, NJ). LiTCA and TMATCA, and TEATCA were prepared by dropwise titration of a solution of 3 N LiOH, TEAOH and TMAOH, respectively, with trichloroacetic acid (100% w / v, 6.1 N) at a pH of 7.0 on ice with constant agitation. The salt was evaporated to dryness under vacuum, washed once with ether and dried.

The oligonucleotides were synthesized in a commercial synthesizer using standard chemistry of cyanoethyl-N, N-diisopropylamino-phosphoramidite (CED-phosphoramidite). The amine ends were incorporated at the 5 'end using the commercially available N-monomethoxytriaminohex-6-yloxy-CED-phosphoramidite. Alternatively, the oligonucleotides were commercially purchased (Midland Certified Reagents, Midland, Tx). Table I shows the oligonucleotides that were used to measure the difference in Td between a wild-type oligonucleotide and a mutant oligonucleotide. The wild type oligonucleotide represents the full duplex and perfectly formed in base pairs and a mutant oligonucleotide represents a mismatch of individual base pair (generally half the oligonucleotide).

TABLE I The oligonucleotides were attached to the tips described herein. In these studies, oligonucleotides were attached to the tips using the aspect described by Van Ness and others, Nucí. Acids Res. 19: 3345, 1991. The oligonucleotide tips contained 0.1 to 1.2 μg / tip of the covalently immobilized oligonucleotide.

(B) Solid Phase Hybridization To label the probe oligonucleotides, the amine oligonucleotides were reacted with reactive amine fluorochromes. The derivatized oligonucleotide preparation was divided into 3 portions, and each portion was reacted with either (a) 20 times the molar excess of Texas red sulfonyl chloride (Molecular Probes, Eugene, OR), with (b) 20 times the molar excess of sulfonyl chloride Lissamine (Molecular Probes, Eugene, OR), or with (c) 20 times the molar excess of fluorescein isothiocyanate. The final reaction conditions consisted of 0.15 M sodium borate (pH 8.3) for 1 hour at room temperature. Unreacted fluorochromes were removed through size exclusion chromatography on a G-50 Sephadex column. A high performance method was developed for measuring the thermodynamic properties of oligonucleotide duplexes. The method allows thousands of solution samples to be explored for their ability to modulate the thermodynamic parameters of the helical to spiral transition of oligonucleotide duplex. This method uses a solid support, which has been designed to fit into a Cetus plate (or the cavity of a plate designated for a 96-cavity PCR format) and requires approximately 40 μL of volume to be completely covered by the liquid . The design of the tip is shown in Figure 1. This tip is also designed to make compatible with the square end of a spring probe that can be used as a binding site in order to arrange the nylon tips in a format of 1x8, 1x12, 4x8, 4x12, or 8x12. An illustration of said device is shown in Figure 2. A member of the oligonucleotide duplex is immobilized on the nylon tip as described by Van Ness and Chem. Nucleic Acids Res. 19: 5143, 1991. A hybridization step is then used to form the oligonucleotide duplexes on a tip. The hybridization step can be carried out in bulk in a single container or individually in the cavities of a plate used for PCR. Therefore, it is possible for each tip of an array of 96 tip members to possess a different oligonucleotide duplex. After the hybridization step, the tips are washed and then placed on a PCR plate mounted on a cycler. In the case of the 1 x 8 or 1 x 12 format, the tips are then moved through a series of cavities each time the temperature is increased by 5 ° C. Typically, the temperature increases are in steps of 5 ° C and the melting period each temperature is from 1 to 5 minutes. For example, tips in a 1 x 12 format are placed in row H at 10 ° C. The thermocycler is then programmed to ramp through 16 steps at 10 minute intervals with 5 ° C temperature increments. The tip arrangement moves from row to row 15 minutes before the temperature increase. In this format, 12 solutions can be studied using two solution plates. In a 96-point format, all solution plates move in and out of the thermocycler at scheduled intervals. Fluorescent probes are commonly used in this format and have little effect on the measured Td values described here. The use of radiolabeled or fluorescent probes allows a wide variety of solutions to be measured, since there is no requirement for optical clarity, in contrast to the case of fusion curves derived by UV spectrometry (displacements of hyperchromicity). Fluorescence is measured with a microtiter plate fluorescence reader, the data is directly imported into a spreadsheet program, such as Excel which then calculates the stability, enthalpy, helical-spiral transition and temperature scale and then graph the results. Typically, a 1 x 12 format that measures 12 solutions at a time can be completed in 1 hour, including data fixation and reduction. For the oligonucleotide oligonucleotide / Td determination of the oligonucleotide tip, the fluorescently labeled oligonucleotide was incubated in several hybridization solutions with a complementary oligonucleotide immobilized at the oligonucleotide tips. From 5 to 5000 ng of the oligonucleotide were hybridized in volumes of 300-400 μl at various temperatures (19-65 ° C) for 5 to 30 minutes. The tips were washed 3 times with one milliliter of the respective hybridization solution, and then once with the respective melting solution at the start temperature of the melting process. The tips in 100 μl of the respective fusion solution were then placed on top of a thermocycler. At intervals of 1 to 5 minutes, the temperature rose 5 ° C and the tip moved to a new cavity of the microtiter plate. The fusion, or dissociation of duplex, was conducted on a temperature scale of 10 ° C to 95 ° C. Fluorescence was measured with a commercial fluorescence plate reader. To calculate the Td, the relative cumulative fluorescent units (RFUs) eluted at each temperature were placed on plates against temperature. The temperature at which 50% of the material was dissociated from the tip is Td or Tm. The helical to spiral transition is defined as the temperature at which a value of a is equal to 0.2 for a duplex of oligonucleotide data (or nucleic acid duplex, containing or not containing a mismatch anywhere in the duplex) to the temperature at which a value for a is equal to 0.8 for the same given oligonucleotide duplex (or nucleic acid duplex). The following Tds were obtained in the hybridizations described below. TABLE II determined. The data indicate that the hibotropic solutions (LiTCA, GuSCN and GuHCI) allow the detection of a mismatch of individual base pairs in a 24-mer and 30-mer probe, while the detection of a base mismatch of individual pairs in standard hybridization solutions (Rapid Hybe, Promega QY or 5x SSC) was not possible A similar experiment was performed for the 24-mer described above in a series of hybridization solutions TABLE III The Td (p) is the T of an oligonucleotide duplex perfectly in base pairs and the Tm (mt) is the Td of an oligonucleotide duplex containing an individual maladjustment. The values shown are for a 24-mer duplex of the sequence described above From the data presented in Table III, the severity factor is directly proportional to the difference between a duplex perfectly in base pairs and a duplex containing a maladaptation. That is, the factor of severity predicts the ability of the solution Hybridization Given to Discriminate Maladaptive Duplexes Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is thus not limited. Those skilled in the art will come up with several modifications that can be made to the described embodiments and that said modifications are intended to be within the scope of the present invention, which is defined by the following claims.

LIST OF SEQUENCES < 110 > Rapigene, Incorporated Garríson, Lori K. Tabone, John C. Van Ness, Jeffrey < 120 > Solid Phase Tips and Uses Related to the Same < 130 > 780068.430PC < 140 > PCT < 141 > 1998-12-29 < 160 > 10 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 39 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide for capture and synthesis of first chain structure used in the gene expression assay < 400 > 1 actactgatc aggcgcgcct tttttttttt ttttttttt 39 < 210 > 2 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 2 gtcatactcc tgcttgctga tccacatctg 30 < 210 > 3 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 3 cagatgggta tcagcaagca ggagtatgac 30 < 210 > 4 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 4 cagatgggta tcaggaagca ggagtatgac 30 < 210 > 5 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild-type and mutant oligonucleotides < 400 > 5 atgggtatca gcaagcagga gtat 24 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 6 atgggtatca ggaagcagga gtat 24 < 210 > 7 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 7 ggtatcagca agcaggag 18 < 210 > 8 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide used to measure the difference in temperature at which half of the nucleic acid duplex molecules are of single strand structure for wild type and mutant oligonucleotides < 400 > 8 ggtatcagga agcaggag 18 < 210 > 9 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Immobilized Oligonucleotide of the Reference Oligonucleotide Representing a Nucleic Acid Duplex < 400 > 9 gtcatactcc tgcttgctga tccacatctg 30 < 210 > 10 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide solution of the reference oligonucleotide representing a nucleic acid duplex < 400 > 10 tgtggatcag caagcaggag tatg 24

Claims (70)

1. A solid phase sample retention tip that can be used in a method for synthesizing or detecting a nucleic acid, comprising: a tip structure that can be connected to a support pin; and a chemical layer covering at least a portion of the tip structure, the chemical layer can be attached to a biomolecule to form a solid phase sample of the biomolecule of the tip structure.

2. The sample retaining tip according to claim 1, wherein the tip structure is removably connectable to the support pin.

3. The sample retention tip according to claim 1, wherein the chemical layer is directly adhered to the tip structure.

4. The sample retaining tip according to claim 1, wherein the tip structure has a partially conical shape with a plurality of slots formed therein.

5. The sample retention tip according to claim 1, wherein the tip structure has a plurality of heat exchange fins therein.

6. The sample retention tip according to claim 5, wherein the tip structure has a partially conical shape.

7. The sample holding tip according to claim 1, wherein the tip structure has an opening thereon sized to receive the support pin therein, the opening having a closed end portion a portion of open end, the open end portion having a generally funnel shape with a transverse cross-sectional area in the direction of the closed end portion.

8. The sample retention tip according to claim 1, wherein the tip structure is nylon 6/6.

9. The sample retention tip according to claim 1, wherein the chemical layer is a polymer having a plurality of amine groups therein that can be attached to the biomolecule.

10. The sample retention tip according to claim 1, wherein the chemical layer is a layer of poly (ethyleneimine).

11. The sample retention tip according to claim 1, wherein the selected chemical layer is covalently attached to the tip structure.

12. A solid phase sample retention assembly for use in a solid phase process for synthesizing or detecting a nucleic acid, comprising: a support pin; a tip structure connected to the support pin; and a chemical layer covering at least a portion of the tip structure, the chemical layer can be attached to a biomolecule to form a solid phase sample of the biomolecule on the tip.

13. The assembly according to claim 12, wherein the support pin is a spring pin.

14. The assembly according to claim 12, wherein the tip structure is removably connected to the support pin.

15. The assembly according to claim 12, wherein the tip structure is a nylon 6/6 member.

16. The assembly according to claim 12, wherein the tip structure has a partially conical shape with a plurality of slots therein.

17. The assembly according to claim 12, wherein the tip structure has a plurality of heat exchange fins therein.

18. The assembly according to claim 16, wherein the chemical layer is a polymer having a plurality of amine groups therein that can be attached to the biomolecule.

19. The assembly according to claim 12, wherein the chemical layer is a layer of poly (ethyleneimine).

20. The assembly according to claim 12, wherein the tip structure has a surface with depressions to provide an increased surface area of the tip structure.

21. The assembly according to claim 12, wherein a first end portion of the support pin has corner portions and a generally polygonal transverse shape, the tip structure has an opening therein defined by a side wall and having a generally circular cross-sectional area, the corner portions frictionally coupling the side wall to retain the tip structure on the support pin.

22. The assembly according to claim 12, wherein the tip structure has an opening therein that removably receives a first end portion of the support pin therein, the opening having a closed end portion and an end portion. open, the open end portion having a generally funnel shape with a reducing cross-sectional area in the direction of the closed end portion.

23. An arrangement of solid phase sample retention assemblies for use in a method for synthesizing or detecting a nucleic acid, comprising: a base; a plurality of support pins connected to the base in a selected arrangement, each support pin having an end portion spaced from the base; a plurality of tip structures connected to the end portions of the support pins; and a chemical layer covering at least a portion of each tip structure, the chemical layer can be attached to a biomolecule to form a solid phase sample of the biomolecule.

24. The arrangement according to claim 23, wherein the support pins are spring pins.

25. The arrangement according to claim 23, wherein the tip structures are removably connected to the support pins.

26. The arrangement according to claim 23, wherein the tip structures are members of nylon 6/6.

27. The arrangement according to claim 23, wherein the tip structure has a partially conical shape with a plurality of slots therein.

28. The arrangement according to claim 23, wherein the tip structure has a plurality of heat exchange fins therein.

29. The arrangement according to claim 23, wherein the chemical layer is a polymer having a plurality of amine groups therein.

30. The arrangement according to claim 23, wherein the chemical layer is a poly (ethyleneimine) layer.

31. The arrangement according to claim 23, wherein the tip structures have a depressed surface thereon.

32. - The arrangement according to claim 23, wherein each tip structure has an opening therein that removably receives an end portion of the respective support pin, the opening having a closed end portion and an open end portion, the end portion open having a generally funnel shape with a transverse area in reduction in the direction of the closed end portion.

33. A combination of solid phase sample retention assembly and microtiter plate for use in a method for synthesizing or detecting a nucleic acid, comprising: a microtitre plate having a cavity shaped to contain a volume of a sample having a biomolecule in it; and a solid phase sample retaining assembly sized to extend at least partially into the cavity, the solid phase sample retaining assembly including: a support pin; a tip structure connected to the support pin, the tip structure being removably removable in the cavity; and a chemical layer covering at least a portion of the tip structure, the chemical layer can be attached to the biomolecule in the solution to form a solid phase sample of the biomolecule.

34. The combination according to claim 33, wherein the support pin is a spring pin.

The combination according to claim 33, wherein the tip structure is removably connected to the support pin and can be placed in the cavity when the tip structure is removed from the support pin.

36. The combination according to claim 33, wherein the tip structure is a nylon 6/6 member.

37.- The combination according to claim 33, wherein the tip structure has a transverse shape that closely corresponds to a transverse shape of the cavity.

38.- The combination according to claim 33, wherein the tip structure has a partially conical shape with a plurality of slots therein.

39.- The combination according to claim 33, wherein the chemical layer is a polymer coating with a plurality of amine groups therein.

40.- The combination according to claim 33, wherein the chemical layer is a layer of poly (ethyleneimine).

41. The combination according to claim 33, wherein the tip structure is frictionally retained on the support pin.

42. The combination according to claim 33, wherein the microtitre plate has a plurality of cavities therein, and further comprises a plurality of the support pins arranged in a selected arrangement, and a plurality of the structures of tips connected to one of the respective support pins to form a disposition of solid phase sample retaining tips that can be placed in the cavities.

43. The combination according to claim 42, further including a base connected to the ends of the support pins separated from the tip structures and the tip structures are substantially coplanar.

44.- A combination of solid phase sample retention tip and microtiter plate for use in a method for synthesizing or detecting a nucleic acid, comprising: a microtiter plate having a cavity shaped to contain a volume of a sample having a biomolecule in it; and a solid phase sample retaining tip removably placed within the cavity, the tip having a tip structure that can be connected to the support pin, while the tip structure is in the cavity, and a chemical layer covering At least a portion of the tip structure, the chemical layer can be attached to the biomolecule in the solution to form a solid phase sample of the biomolecule on the tip.

45. - The combination according to claim 44, wherein the tip structure is a nylon 6/6 member.

46. The combination according to claim 44, wherein the tip structure has a transverse shape that substantially corresponds to a transverse shape of the cavity.

47. - The combination according to claim 44, wherein the tip structure and the cavity have transverse, partially conical shapes.

48. The combination according to claim 44, wherein the tip structure has a partially conical shape with a plurality of slots therein.

49.- The combination according to claim 44, wherein the chemical layer is a layer of poly (ethyleneimine).

50.- A method for manufacturing a solid phase sample retention tip for use in solid phase molecular biology processes, comprising the steps of: forming a substrate material such as a tip structure that can be attached to a pin of support; covering at least a portion of the substrate material with a chemical layer that can be attached to a selected biomolecule to form a solid phase sample of the biomolecule; and joining the chemical layer to the substrate material.

51. The method according to claim 50, wherein the step of attaching the chemical layer to the substrate material includes covalently attaching the chemical layer to the substrate material.

52. - The method according to claim 50, wherein the chemical layer is a polymer having a plurality of amine groups therein that can be attached to the biomolecule, and the step of joining includes covalently bonding the polymer of substrate material .

53. The method according to claim 50, wherein the chemical layer is a poly (ethyleneimine) and the substrate material is a nylon 6/6 material, and the step of joining includes covalently joining the poly (ethyleneimine) to nylon 6/6.

54. The method according to claim 50, further comprising the step of attaching the tip structure to the support pin.

55. The method according to claim 54, wherein the joining step of the tip structure includes removably joining the tip structure to one end of the support pin. 56.- The method according to claim 50, further comprising the step of providing a base, a plurality of support pins and a plurality of the tip structures with the chemical layer thereon, joining the support pins to the base in one arrangement, and joining the tip structures to a respective pin of the plurality of support pins to form a disposition of solid phase sample retaining tips that are separated from the base. 57.- A method for forming a solid phase sample of a biomolecule, comprising the steps of: submerging a portion of a tip assembly in a solution having the biomolecule therein, the tip assembly having a portion of substrate and a chemical layer on the substrate portion, the chemical layer can be attached to the biomolecule; allowing the biomolecule to bind to the chemical layer to form a solid phase sample of the biomolecule in the tip assembly; and remove the tip assembly from the solution after the biomolecule has joined the chemical layer. 58. The method according to claim 57, wherein the solution is contained in a cavity of a microtiter plate, and the step of submerging includes placing a portion of the tip assembly in the cavity. 59. The method according to claim 57, which further includes the steps of performing a molecular biology process on the tip assembly after the biomolecule has been attached to the chemical layer. The method according to claim 57, further comprising the step of storing the solid phase tip assembly in a retention member after the biomolecule has been attached to the chemical layer. 61.- The method according to claim 60, wherein the retention member is a microtitre plate having a cavity therein, and the step of storing includes placing the tip assembly in the cavity after the biomolecule has been attached to the chemical layer, and placing the plate of microtitre and tip assembly as a unit in a storage site. 62. The method according to claim 57, wherein said biomolecule is either a nucleic acid or an amino acid polymer. 63. The method according to claim 62, wherein the nucleic acid is an oligonucleotide having one end attached to the chemical layer and a free end. 64.- The method according to claim 63, wherein the oligonucleotide free end comprises an oligo (dT) sequence. The method according to claim 64, further comprising the step of attaching poly (A +) RNA to said tip assembly, wherein the poly (A +) portion of said poly (A +) RNA is linked to the oligonucleotide oligo (dT) sequence. 66. The method according to claim 65, further comprising the step of synthesizing cDNA of said bound poly (A +) RNA. 67.- The method according to claim 57, wherein the biomolecule is an avidin molecule. The method according to claim 67, further comprising the step of attaching an oligonucleotide to the tip assembly, wherein said oligonucleotide comprises at least a portion of biotin, and wherein the biotinylated oligonucleotide is attached to the molecule of avidin. 69.- The method according to claim 59, wherein the molecular biology process is selected from the group consisting of cDNA synthesis, polymerase chain reaction, preparation of a collection of subtracted cDNA, synthesis of differential probes, minisequencing of solid phase, oligonucleotide ligation assay, and amplified fragment length polymorphism analysis. 70. The method according to claim 62, wherein the amino acid polymer is an antibody.