WO2003066878A2 - Nucleic acid analysis using layered expression scanning - Google Patents

Abstract

Provided herein are methods of producing duplicates of nucleic acid samples, such as Northern blots, Southern blots, and sequencing gels, and methods of analyzing nucleic acids on such duplicates. Also provided are kits for using in producing duplicates of nucleic acid samples, and reagents for use in these kits and methods.

Description

NUCLEIC ACID ANALYSIS USING LAYERED EXPRESSION SCANNING

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/353,407, filed February 1, 2002, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure is directed to methods, devices, arrays, and kits for identifying and analyzing large numbers of nucleic acids in a sample. The disclosure further relates to using these methods, devices, arrays, and kits to analyze functions and roles of nucleic acids, such as their functions and roles in disease, and to correlating the presence, absence, or quantity of a combination of nucleic acids with particular diseases, prognoses, or responses to therapies.

BACKGROUND OF THE DISCLOSURE Understanding the molecular events that characterize tumorigenesis is a key step to designing diagnostic, prognostic, and therapeutic agents. However, tumorigenesis is a multistep, complex process, usually involving de-regulation of several genetic pathways. Therefore, in recent years, great effort has been directed toward molecular profiling of cancer, i.e., global measurements of gene expression patterns that characterize the different phases of tumor progression (Schena, BioEssays 18: 427-431, 1996; Chee et al, Science 21 A: 610-614, 1996; DeRisi et al, Nature Gen. 14: 457-460, l996; Luek g et al, Anal. Biochem. 270: 103-111, 1999; Paweletz et α/. Oncogene 20: 1981-1989, 2001). Microarray and proteomics studies analyzing thousands of genes have yielded long lists of dysregulated genes in cancer, each gene with a potential clinical value (Clarke et al, Biochem. Pharmacol. 62: 1311-36, 2001; Liotta et al. JAMA 286: 2211-4, 2001). However it is often important to validate and/or confirm these results in a separate, independent experiment. Therefore, there is an increasing need for a rapid, quantitative method to validate the differential expression of genes identified by high throughput technologies. Several approaches have been used, including immunohistochemistry (IHC) or Western blots at the protein level, and Northern blotting, in situ hybridization (ISH) or RT-PCR at the RNA level. In situ techniques such as ISH or IHC yield results that are difficult to quantitate, and background signals increase this problem. Blotting techniques present the advantage that signals are easy to quantitate, the size of the bands serves as a control for the specificity of the signal, and no amplification bias is introduced in the analysis. However, blotting involves cumbersome techniques, and usually only one gene is analyzed at a time. Northern blotting still remains the most reliable method for analyzing the expression level of a specific gene in a sample (Southern, Trends Biochem. Sci. 25:585-588, 2000). No amplification step is required to assess the gene expression level. The signals are easy to quantitate, an internal housekeeping control can be used, and the size of the band serves as a control for the specificity of the signal. However, it is a cumbersome technique requiring different steps, and usually only one gene is analyzed in each experiment.

Layered Expression Scanning (LES) was recently developed and allows the movement of molecules through several layers of membrane (Englert et al, Cancer Res, 60(6):1526-1530, 2000). However, this technology has not previously been optimized for use with nucleic acid transfer and analysis.

It would be desirable to have high throughput approaches for detecting, identifying and comparing large numbers of nucleic acids that are relatively inexpensive and readily permit the capture, organization, and analysis of the data generated thereby.

SUMMARY OF THE DISCLOSURE

The present disclosure provides ultrasensitive blotting techniques for nucleic acid analysis based on layered expression scanning. These techniques allow the generation of several replicates of an original nucleic acid sample, therefore increasing the output for both Northern and Southern blotting and sequence analysis, and enabling replicate analysis of nucleic acids from tissue sections. In certain embodiments, replicate membrane techniques are provided that permit multiple copies of nucleic acid samples, particularly including nucleic acid gels, to be produced for subsequent analysis.

In another embodiment, a direct nucleic acid capture technique is provided that permits specific nucleic acid targets to be directly analyzed on membranes. In some embodiments, specific coating of individual membrane layers allows the simultaneous hybridization of target nucleic acids to the corresponding layers during the transfer, further increasing the output of the analysis.

In yet other embodiments, nucleic acids are amplified prior to or after transfer of a sample to the membranes. These embodiments permit even minute amounts of nucleic acids to be analyzed.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic representation of two blotting approaches disclosed herein: Fig. 1A illustrates generation often replicates from one sample for nucleic acid analysis (Northern or

Southern blotting); Fig. IB illustrates hybridization of nucleic on gene-specific coated membranes during the transfer.

Figure 2A is an oblique view of an apparatus shown transferring nucleic acids from a tissue section onto a membrane stack. FIG. 2B is a front view of an assembled contact transfer stack, prepared for transfer in the apparatus illustrated in FIG.2A.

Figure 3 is a longitudinal sectional view of an individual membrane according to one provided embodiment.

Figure 4 is a schematic drawing, illustrating direct capture. Figure 5 is a schematic drawing, illustrating indirect capture.

Figure 6 is a perspective view of a representative framed membrane stack.

Figure 7 is a front elevation view of a single framed membrane.

Figure 8 is a sectional view of the single membrane taken along line 115-115 of FIG. 7. Figure 9 shows a series of duplicated Northern Blots, illustrating staining of total RNA on

10 T-T layers. The ten replicates were generated from a gel that was loaded with 15 μg of MDA453 cell line total RNA.

Figure 10 shows a series of blots that correspond to GAPDH hybridization signals on layers 2, 4, 7, and 8 (of 10 original layers). The duplicate Northern blots were generated from a gel that was loaded with 15 μg of Osteosarcoma MG-63 cell line total RNA.

Figure 11 shows a comparison of GAPDH hybridization signals on T-T layer # 2 (of 10 Northern blotting replicates) and one nitrocellulose membrane generated using conventional Northern blotting. The two experiments were performed in parallel, starting with 15 μg of Osteosarcoma MG- 63 cell line RNA. Figure 12 shows hybridization of RT-PCR products with membranes, where the membranes were coated with specific capture molecules prior to transfer, thus enabling different membranes to capture different transferred molecules.

FIG. 12A shows the transfer of β globulin RT-PCR product (370 bp) through blocked, beta globulin-coated, blocked, actin-coated, and blank layers. A specific hybridization signal is seen on the β globulin-coated layer, and no background signal on the actin-coated layer.

FIG. 12B shows blots from an acrylamide gel in which three lanes were loaded with RT- PCR products, as follows: lane 1: c-jun (479 bp) + c-myc (409 bp); lane 2: c-jun (479 bp); and lane 3: c-myc (409 bp). The RT-PCR products were transferred through layers that were coated with: c- myc, c-jun, and both c-myc and c-jun. Specific hybridization of each product is seen on the expected layers.

Figure 13 shows (in the leftmost panel) the separation of MUC1 cDNA restriction fragment (0.7 kb) and linearized plasmid DNA (3kb) on a 0.8% agarose gel. Following transfer from the gel to 10 membrane layers, MUC1 cDNA was detected on membrane layers 2 and 5, and plasmid DNA was detected on membrane layers 4 and 7, in each case by hybridization to a sequence specific probe. Figure 14 shows a schematic diagram of one embodiment of the Spin Transfer

Amplification LES protocol.

Figure 15 is a scanning electron micrograph showing a representative "track-etched membrane" (a/k/a "screen membrane").

Figure 16 is a scanning electron micrograph showing a representative "depth" or "tortuous pore" membrane.

Figure 17 is a series of RNA replica blots, illustrating the transfer of nucleic acids into stacks of membranes to make replicate blots of a starting sample in a gel. FIG 17A shows total RNA staining on ten membrane layers generated from a gel loaded with 15 μg of MDA-MB-453 cell line RNA. FIG 18B shows GAPDH signal on membranes from layers 2, 4, 7 and 8 of a stack of 10 membranes, transferred from a gel loaded with 15 μg of Osteosarcoma MG-63 cell line RNA.

Figure 18 is a series of RNA blots. FIG 18A shows two blots comparing GAPDH hybridization signal on a layered array membrane (left panel) with that from a standard nitrocellulose membrane (right panel). Both experiments were performed in parallel using 15 μg of Osteosarcoma MG-63 cell line RNA. FIG 18B shows three images of the same layered array membrane, which has been probed with GAPDH probe (1.3 kb) (left panel), stripped as described in the text (middle panel), and reprobed with beta actin probe (2.1 kb) (right panel).

Figure 19 is a series of four images of RNA blots, illustrating the uniformity and reproducibility of the layered array system. The four panels show hybridization to GAPDH, HPV-18 E6/E7, PCNA, and cdc2 (respectively). The gel used to transfer nucleic acids to the membrane stack was loaded with 20 μg of HeLa cell line total RNA per lane.

SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is a 5' beta microglobin primer.

SEQ ID NO: 2 is a 3' beta microglobin primer.

SEQ ID NO: 3 is a 5' actin primer.

SEQ ID NO: 4 is a 3' actin primer. SEQ ID NO: 5 is a 5' HPRT primer.

SEQ ID NO: 6 is a 3' HPRT primer.

SEQ ID NO: 7 is a 5' c-myc primer.

SEQ ID NO: 8 is a 3' c-myc primer.

SEQ ID NO: 9 is a 5' c-fos primer. SEQ ID NO: 10 is a 3' c-fos primer.

SEQ ID NO: 11 is a 5' c-jun primer.

SEQ ID NO: 12 is a 3' c-jun primer.

DETAILED DESCRIPTION I. Explanation of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:

"Affinity" means the chemical attraction or force between molecules.

"Amplification" when used in reference to nucleic acids means any one of a variety of Techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Other examples of amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription- free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134). In situ amplification refers generally to an amplification reaction that is carried out in the place that the template nucleic acid occurs (rather than in solution in a reaction vessel), for instance, in a tissue section. Other examples of in situ amplification include what is referred to herein as "in stack-ό" and "in membrane-o" (occurring on a membrane or stack of membranes to which the nucleic acid template has been transferred) and "in gel-o" (occurring in a gel, such as an electrophoretic gel) amplification.

"Array" means two or more. "Biological sample" or "sample" means any material containing biomolecules, whether solid, liquid or gas, including, e.g., organs, tissues, bodily fluids, cells in suspension or pelleted, cell or tissue extracts, and gels or other materials used to separate and/or immobilize biomolecules (e.g., agarose gels and polyacrylamide gels).

"Biomolecules" are molecules of biological origin, which are typically produced by, obtained from, excreted by, secreted by or derived from living organisms (including microorganisms, viruses, plants, animals, and humans). The term biomolecules includes, without limitation, peptides, proteins, glycoproteins, nucleic acids, fatty acids, and carbohydrates.

"Capacity" means the ability to receive, hold, or absorb biomolecules from a sample.

"Captor" means a molecule, such as an antibody or nucleic acid {e.g., DNA) probe, that is anchored to a membrane and has an affinity (such as a specific affinity) for a biomolecule.

"cDNA" refers to a DNA molecule lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA may be synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. "Counter-ligand staining" is intended to refer to any detection technique that detects the presence of ligand that is not bound to a protein of the biological sample, and thus reveals (as, for example, by an absence of staining, etc.) the presence of ligand that is bound to a protein of the biological sample "Detector" means a molecule, such as an antibody or DNA probe, that is free in solution (i.e. not anchored to a membrane) and has an affinity for one of the sample components.

"Direct capture" means the conjugation or binding of a biomolecule directly onto the surface of the membrane without the aid of a captor, such as a nucleic acid probe or antibody or the like.

"DNA" is a long chain polymer that contains the genetic material of most living organisms (the genes of some viruses are made of ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases (adenine, guanine, cytosine, and thymine) bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term "codon" is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

"EST" (Expressed Sequence Tag) is a partial DNA or cDNA sequence, typically of between 500 and 2000 sequential nucleotides, obtained from a genomic or cDNA library, prepared from a selected cell, cell type, tissue or tissue type, organ or organism, which corresponds to an mRNA or genomic fragment represented by or found in that library. An EST is generally a nucleic acid molecule sequenced from, and shorter than, the cDNA or genomic DNA from which it is obtained. "Fluorophore" refers to a chemical compound, which when excited by exposure to a particular wavelength of light, emits light (i.e., fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or "color." Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 λ. Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-690 λ.

Examples of fluorophores that may be used are provided in U.S. Patent No. 5,866,366 to Nazarenko et al, and include for instance: 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3 -(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6- dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron .RTM. Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

Other suitable fluorophores include GFP (green fluorescent protein), Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivatives thereof. Other fluorophores known to those skilled in the art may also be used. "High throughput genomics" refers to application of genomic or genetic data or analysis techniques that use microarrays or other genomic technologies to rapidly identify large numbers of genes or proteins, or distinguish their structure, expression, or function from normal or abnormal cells or tissues.

"Hybridization" refers to an interaction between nucleic acid molecules that are complementary to each other. Hybridization is based on hydrogen bonding, which includes Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleotide units. For example, adenine and thymine are complementary nucleobases that pair through formation of hydrogen bonds. "Complementary" refers to sequence complementarity between two nucleotide units. For example, if a nucleotide unit at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide unit at a certain position of a DNA or RNA molecule, then the nucleotides at those positions are complementary to each other. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units which can hydrogen bond with each other.

"Specifically hybridizable" and "complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. An oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), chapters 9 and 11, herein incorporated by reference

"Indirect capture" means the conjugation or binding of a target biomolecule onto a captor nucleic acid or the like which in turn is bound to the surface of the membrane. Thus, with indirect capture the target biomolecule is not directly conjugated to the membrane.

"Label" refers to detectable markers or reporter molecules, which can be attached for instance to a specific biomolecule (e.g., a nucleic acid). Typical labels include fluorophores, radioactive isotopes, ligands, chemiluminescent agents, metal sols and colloids, and enzymes. Methods for labeling and guidance in the choice of labels useful for various purposes are discussed, e.g., in Sambrook et al, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory Press (1989) and Ausubel et al, in Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987).

"Membrane" means a thin sheet of natural or synthetic material that is porous or otherwise at least partially permeable to biomolecules. "Microarray" is an array comprising addressable locations that is miniaturized so as to require microscopic examination for (e.g., visual) evaluation.

"Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, and encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. "Stack" refers to a plurality (e.g, 2, 3, 4, 5, 8, 10, 15, 20, 50, 100 or more) of adjacent substrates, whether oriented horizontally, vertically, at an angle, or in some other direction. The substrates (e.g., membranes) may be spaced or touching, for example contiguous.

"Subject" refers to living, multicellular vertebrate organisms, a category that includes both human and veterinary subjects for example, mammals, birds, and particularly primates. "Two-dimensional relationship" refers to the physical location of two objects in relation to each other in two dimensions of space. The two dimensions are usually defined by some surface of reference, for instance one surface of a three-dimensional object such as a block or slice of a solid substance. For instance, two bands in a gel are in a defined two dimensional relationship to each other, in that the two bands can be located and identified, relative to each other, in a two dimensional space that is defined by the face of the gel. Likewise, the nucleic acid species of those two bands are in a defined two-dimensional relationship to each other. In various embodiments herein, the sample is a sample that contains nucleic acids (or nucleic acid species) that are in defined two-dimensional relationships to one another, for instance based on their positions within a polymerized matrix (such as a gel), on a membrane, on several membranes within a stack, within a tissue section, in the features of an array or microarray, in wells of a microtiter plate (which is in and of itself a form of array), and so forth. Methods are provided wherein nucleic acids in a sample having a defined two-dimensional architecture (wherein nucleic acids are maintained in defined two-dimensional relationships relative to each other) are transferred from the sample to membranes of a stacked array of membranes.

Particularly, in such methods the two-dimensional architecture is substantially maintained during the transfer. Thus, nucleic acids captured on the membranes are captured in positions that correspond to the positions (in the two dimensions defined by the face of the sample and the surface of the stack to which the sample was applied) they held relative to each other in the starting sample. It is understood, however, that the maintenance of position is relative rather than absolute, and the replicates produced by methods described herein are not limited to exact identical copies of the two- dimensional architecture of the starting sample.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

II. Overview of Several Specific Embodiments

Disclosed in a first embodiment is a method of detecting one or more nucleic acids in a sample (e.g., a RNA/Northern gel, a DNA/Southern gel, a nucleic acid sequencing gel, a tissue section, or a nucleic acid array), which method involves providing a stack of at least two membranes; contacting the sample to the stack produce a loaded stack; maintaining the loaded stack under conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack, and allow capture of at least a portion of the nucleic acids on the membranes; and detecting at least one nucleic acid on one or more of the multiple membranes, wherein the nucleic acids in the sample are in defined two dimensional relationships relative to each other, and wherein the portion of the nucleic acids captured on the membranes substantially maintains at least a subset of those defined two dimensional relationships. Examples of this method further involve amplifying nucleic acids in the sample, for instance, prior to contacting the sample to the stack or after contacting the sample to the stack. In another embodiment, the method is a method of making multiple substantial copies of the nucleic acid content of the sample, and the multiple membranes are the multiple substantial copies.

In further example methods, capture of at least a portion of the nucleic acids on at least one of the membranes is by direct capture, or by indirect capture. Membranes in various provided methods each comprise a porous substrate having a thickness of less than 30 microns. The porous substrate may comprise, for instance, a material selected from the group consisting of polycarbonate, cellulose acetate, polyester, polyethylene terephthalate, polyethelyle, polypropylene, and mixtures thereof.

Optionally, membranes can comprise a material (or mixture of materials) for increasing affinity of the membrane to nucleic acids. In certain examples, the material for increasing affinity of the membrane is coated on the at least one membrane, in other examples, it is mixed into the substrate. By way of example, the material for increasing affinity can be selected from the group consisting of nitrocellulose, poly-L-lysine, and mixtures thereof. Alternatively, the material for increasing affinity comprises a nucleic acid-specific captor. In particular envisioned embodiments, the porous substrate of the membranes comprises a polycarbonate substrate, and the material for increasing affinity comprises nitrocellulose.

A further embodiment is a method in which detecting the nucleic acids involves separating at least one membrane from the stack; and detecting at least one nucleic acid on the one or more of the separated membranes. In yet a further embodiment, the conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack comprise passing a transfer liquid through the membranes. Optionally, the conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack comprise providing a wick that facilitates movement of the nucleic acids through the stack of membranes in a desired direction of movement. In any of the provided methods, the stack of membranes can include 3 or more membranes, for instance 5 or more membranes, or even 10 or more membranes. Optionally, at least two of the membranes within a stack have differential binding affinities for nucleic acids.

In specific embodiments, amplifying the nucleic acids in the sample comprises in situ amplification of at least one nucleic acid molecule prior to applying the sample to the stack. For instance, this may involve in membran-o amplification or in gel-o amplification of at least one nucleic acid molecule.

Also described herein is a method of making multiple substantial replicate blots of a nucleic acid content of a sample (e.g., a RNA/Northern gel, a DNA/Southern gel, a nucleic acid sequencing gel, a tissue section, or a nucleic acid array), which method involves providing a stack of at least two membranes, wherein the membranes permit nucleic acids applied to a surface of the stack to move through the membranes, while capturing at least a portion of the nucleic acids on each of the membranes; and applying the nucleic acid sample to the stack, under conditions that allow the membranes to capture at least a portion of the nucleic acids from the sample, thereby making multiple substantial replicate blots of the nucleic acid content of the sample. Examples of this method further involve amplifying the nucleic acids prior to applying the nucleic acid sample to the stack, for instance before or after applying the nucleic acid sample to the stack. In examples of the method, at least a portion of the nucleic acids are captured on at least one membrane by direct capture, or by indirect capture. Optionally, at least two of the membranes within the stack have differential binding affinities for nucleic acids. Optionally, nucleic acids in the sample are amplified, for instance in situ amplified prior to applying the sample to the stack.

Also provided are methods that further involve detecting one or more nucleic acids of interest on at least one of the multiple substantial replicate blots. For instance, detecting one or more nucleic acids of interest in some examples involves exposing a plurality of the multiple replicate blots to at least one detector molecule. Optionally, methods further involve separating the multiple membranes prior to detecting the nucleic acids of interest.

Other provided embodiments are methods in which the nucleic acids in the sample are separated on a solid support, and wherein the method comprises contacting the resultant separated sample to the stack. For instance, the nucleic acids are in some instances separated on a gel. In specific examples, separation of the nucleic acids involves electrophoresis, for instance electrophoresis of a poly- A RNA sample or a total RNA sample. By way of example, in specific examples less than about 1 μg of poly- A RNA is loaded into a well of the gel for separation. In other examples, less than about 0.5 μg of poly-A RNA, less than about 0.1 μg of poly-A RNA, or less than about 0.01 μg of poly-A RNA is loaded into a well of the gel. In other examples, a sample comprising more than about 5 micrograms of total RNA is loaded into a well of the gel. In other examples, the sample comprises about 10 to about 20 micrograms of total RNA is loaded into a well of the gel. Nucleic acids in these embodiments are optionally amplified prior to contacting the stack to the separated sample. For instance, in specific examples of the method, the nucleic acids are amplified within the solid support.

In any of the described embodiments, the membranes can have a high affinity but a low capacity for nucleic acids. Each membrane in a stack is optionally less than about 30 microns thick, for instance about 8 to 10 microns thick. In any of the described embodiments, at least one side of the membranes is treated to increase specific binding of the nucleic acids. Optionally the membranes used in the described methods are in a frame, the frame being mounted to the periphery of the membranes, wherein the frame defines a channel for passing fluids or air away from the space intermediate between the membrane and an adjacent membrane.

In examples of the disclosed methods, the stack includes a plurality (for instance, at least 2, 5, 7, 8, 9, 10, 12, 15, 20, 25, or more) of porous substrates each having a thickness of less than 30 microns, for instance about 8-10 microns. In some embodiments, the porous substrates include polycarbonate, cellulose acetate, or mixtures thereof. The membrane substrates in some embodiments include a material for increasing the affinity of the membrane to the nucleic acids, which material may be coated on (one side, the other, or both) one or more of the membranes. By way of example, this material is in some specific examples nitrocellulose, poly-L-lysine, and mixtures thereof, or a nucleic acid specific ligand (specific either for a target nucleic acid or for nucleic acids in general).

In particular examples of the described methods, the porous substrate includes a polycarbonate substrate and nitrocellulose.

In various embodiments of the disclosed methods, the sample is a gel, for instance a RNA Northern gel, a DNA/Southern gel, or a sequencing gel. In other embodiments, the sample is a tissue samples such as thin section slices (e.g., archival or frozen tissue samples), a tissue array (e.g., a tissue microarray), nucleic acid prints on filter paper, or an environmental sample. This disclosure further provides kits. Some of the kits are kits for replicating a pattern of nucleic acids from a sample; these kits contain a plurality of membranes, each having a coating on its upper and/or lower surfaces to increase specific binding of a target biomolecule; a quantity of transfer buffer (e.g., SSC or TBE); and optionally a fluid impervious enclosure. Also provided are kits for uniquely visualizing a desired predetermined nucleic acid if present in a biological sample, which kits include a plurality of membranes, each having a specific affinity for at least one nucleic acid, and at least one detector species, adapted to detect the desired predetermined nucleic acid if bound to the membranes.

III. Nucleic Acid Layered Expression Scanning The most widely used method for identifying and measuring nucleic acids is gel electrophoresis, a collection of techniques for separating or resolving nucleic acids in a mixture under the influence of an applied electric field based on (usually) the difference in their size and/or charge. Electrophoretic separation is most commonly performed using porous polymer gels. During one- dimensional electrophoresis, a mixture of nucleic acids is applied to a gel and exposed to the flow of an electric current. Since smaller nucleic acids migrate faster through the gel than larger ones, separation based on their size is achieved.

According to specific methods provided herein, nucleic acids that have been electrophoretically separated on a gel, or via chromatography, etc., are transferred from the gel onto and into a stack of membranes. Examples of such membranes are membranes that are constructed and chemically treated to have a high affinity but low capacity for nucleic acids. Suitable membranes and methods for their construction and preparation are described herein. The use of such membranes allows the creation of multiple replicates of the nucleic acid content of the gel.

One of the disclosed systems enables the generation of replicates (non-specific or replicate capture of a proportion of all the material in the nucleic acid sample by each membrane). In certain embodiments, individual membranes may be coated in such a manner as to have differential affinity for nucleic acids (for example, membrane 2 in a stack might have a lower binding affinity than membrane 8). For instance, such variations in binding affinity could be used to increase consistency in binding from membrane to membrane within a stack. Another of the disclosed systems involves pre-coating at least some of the membranes in the stack with a capture molecule, so that nucleic acids in the sample will be specifically captured on different layers according to the specific hybridization that is taking place between the sample and the capture molecule on each membrane (specific capture). In one embodiment, referred to as direct transfer, the membranes are then incubated with a detector molecule or mixture or cocktail of such, to assist in and permit detection and/or analysis of nucleic acids on the membranes. The membranes are generally separated one from another prior to such incubation. Detector molecules/ligands can be any of a number of molecules that have binding specificity for a target nucleic acid of interest, and include molecules that bind or hybridize to nucleic acids (e.g., nucleic acid probes or specific binding proteins or fragments thereof) etc. While in certain embodiments each membrane has essentially the same pattern (two-dimensional architecture of nucleic acids in relation to each other) of nucleic acids bound to it, different combinations of such nucleic acids can be detected on each membrane due to the particular detector or cocktail of detectors selected to probe the particular layer. In certain embodiments, a membrane will be incubated in the presence of a single detector molecule, or a cocktail of different detector molecules of the same class. Alternatively, a membrane may be incubated with different classes of detector molecule(s). Where mixtures or cocktails of detector molecules are employed, the mixtures are optionally formulated so that no two detectors bind overlapping or adjacent nucleic acid bands. Thus, for example nucleic acid bands that are too close together to be discriminated on a single membrane may be detected on separate membranes. Particular embodiments are a method and a kit for identifying (i.e., detecting, annotating, and/or characterizing) groups of nucleic acids that have been separated by gel electrophoresis. In one example, such a kit generally comprises the following components: (i) a stack of membranes upon which the nucleic acids are transferred, (ii) hybridization probes, for instance one for each of the membranes, and (iii) other reagents including nucleic acids transfer buffer and hybridization reagents. The kit may also include software that allows the user to analyze and manipulate the images produced so as to yield a "nucleic acid image" of the sample being tested and compare it to images from other samples in a database. Alternatively the software may be acquired or accessed independent of the kit. In a specific embodiment, a membrane stack comprises a plurality of membranes adapted to be stacked atop one another such that they may later be separated from one another.

According to the method of a particular embodiment, nucleic acids that have been electrophoretically separated on gel are transferred from the gel through a membrane stack. This allows the creation of multiple replicate blots of the nucleic acids content of the gel. The membranes are then separated, and each is incubated with one of the unique detector molecules, e.g., hybridization probes. The probes employed are labeled or otherwise detectable using any of several techniques. This produces unique spot/band patterns on each of the membranes. The membranes with unique patterns are then scanned or digitally imaged using an imaging instrument so that the density of the features may be calculated, compared to other samples, and displayed on a computer using software, as described herein.

One advantage of specific embodiments provided herein is that they provide an additional dimension of nucleic acid separation for a sample. The layered membranes provide a cost-effective tool for selecting groups of hybridization probes (or other detection molecules) that can be used to detect subsets of nucleic acids on the same membrane. Once selected, these detector combinations can be packaged in a kit and used repeatedly for the controlled analysis of nucleic acids displayed on stacked membranes. Since 5-10 or more replicates or copies can be generated from a single gel and ten or more detectors can be applied to each membrane, several thousand different nucleic acids can be identified from a single gel according to methods described herein.

Since ligands can be used to detect epigenomic modification of nucleic acids (e.g. methylation), the present disclosure can also be employed to identify, measure, quantify, and/or study such modifications.

In some examples, only about 10 membranes are used in each transfer stack. It has been observed that membranes 9 and 10 sometimes show a decrease in the signals, particularly when relatively low amounts of nucleic acid is loaded into the stack from the gel. More total RNA, but a more homogeneous distribution of the signals, is seen throughout all 10 layers if about 10 to 25 μg, for instance about 20 μg, of total RNA is used. Loading 5 μg of polyA or messenger RNA (which would correspond roughly to the amount found in a sample containing about 100 μg of total RNA) without any amplification of the nucleic acids yields consistent signals in the first 4-5 membranes. It is believed that the rRNA in total RNA samples serves as an effective carrier for the RNA transfer. Similarly, rRNA or another relatively non-specific nucleic acid can be used as a carrier in low- messenger RNA procedures to increase the migration of the RNA into the membrane stack.

Alternatively, amplification (global or specific for one or more target nucleic acids) can be used to increase the relative amount of RNA in a sample before it is analyzed on the membranes from a transfer stack. For instance, nucleic acids can be amplified before the nucleic acids are separated on a gel, within a tissue sample (traditional in situ) before the nucleic acids are transferred into a membrane stack, within a gel but before transfer the membrane stack (in gel-o), or on the membranes after transfer (in membrane-o or in stack-ό). For coating membranes for use in specific capture (indirect capture) hybridization methods, the following variables have been examined: DNA concentration; denatured vs. non-denatured DNA; different solvents: H₂0, TBE, SSC, each with or without SDS; cross linking, non-cross linking, or baking the load membranes; and direct versus indirect (using DNA in excess solution) at various different temperatures. Though optimal conditions are set forth herein for transferring nucleic acids into a membrane stack, some variations are tolerated by the system. In particular, the membranes in various embodiments are not pre-treated, or are pre-treated with water or different concentrations of TBE or SSC (either with or without methanol). Various transfer systems (including conventional capillary transfer, upside down transfer, electrotransfer, and direct contact transfer both with and without heat, with and without microwaving, and with and without pressure) can be employed to transfer nucleic acids using methods described herein. Though specific buffer concentrations are provided, such as specific SSC concentrations, these too can be varied. Optionally, the nucleic acid samples can be subjected to hydrolysis (e.g., a short step using very diluted sodium hydroxide (0.005M), for 5 minutes or so) prior to transfer.

₍ Similarly, various means of hybridization can be used to analyze membrane replicates produced using methods described herein. These include, for instance, coverslip hybridization (with or without employing a steam chamber) at various different temperatures from about 4 °C to about 70 °C; transparency (plastic sheet) hybridization (with or without employing a steam chamber) at various different temperatures from about 4 °C to about 70 °C; rotating tubes, similarly at various different temperatures; slot blotting (with or without employing a steam chamber) at various different temperatures from about 4 °C to about 70 °C; and specific molecule capture (as described more fully herein) (with or without employing a steam chamber) at various different temperatures from about 4 °C to about 70 °C.

Further variables that have been examined, include time of hybridization (from 15 minutes to overnight); purification of the loaded PCR products; solution in which the DNA is suspended when used to coat the membranes (PCR buffer vs. different concentrations of SSC buffer); probe concentration; membrane coating DNA concentration; the presence of urea, formamide, or salts in hybridization solution; with and without prehybridization; leaving the membranes stacked during hybridization; and membranes differently coated with nitrocellulose or another coating material (for instance, where the coating concentration is 0.01, 0.1, 1, 10 fold compared to the normal concentrations used to coat the membranes).

Optionally, membrane blots produced with the methods described herein can be stripped and re-probed with, for instance, a different hybridization probe. Stripping is generally carried out as is known to those of ordinary skill in the art.

In one particular embodiment for directly transferring RNA from a gel into a stack often membranes, the following broad overview procedure is used:

Optionally individual membranes are labeled (e.g., with a permanent black marker), to assist in identification of individual layers and orientation. Non-stacked membranes are pretreated in 10 X TBE, at room temperature, shaken for 30 minutes, then briefly washed in 20X SSC and assembled into a stack.

By way of example, for sample preparation a standard 1% agarose RNA gel is run, using 15 μg total RNA per lane. Optionally, the gel is subjected to hydrolysis, 0.005 M Sodium Hydroxide, 1.5 M Sodium Chloride, room temperature, shaking, 10 minutes; then neutralized (0.5 M Tris-HCl pH 8.0, 1.5M Sodium Chloride, room temperature, shaking, 10 minutes). It is then re-equilibrated by incubating in 20 X SSC, 5 minutes. The sample is transferred from the gel into a stack of membranes using capillary transfer, overnight at room temperature, using the following tower assembly (from top to bottom):

Standardized weight: 7 g / cm²

Transfer paper: Gel Blot Paper (Schleicher & Schuell), GB004 Optional nitrocellulose trap

Membranes Gel 20 X SSC transfer buffer (reservoir at the bottom, which wicks up through the membrane). The stack is disassembled and the transferred nucleic acids UV-crosslinked to the individual membranes. Pre-hybridization is carried out in: 6 X SSC, 0.5% SDS, 10 μg/ml salmon sperm DNA, 5 X Denhardt's. Membranes are then hybridized with the desired probe using 1-lOk cpm /μl hybridization solution. Hybridized membranes are washed and exposed to radiographic or phosphoimager screen using standard techniques.

IV. Transfer Modes

Provided herein are multiple methods for transferring nucleic acids from a substantially two- dimensional gel or tissue section into one or more thin membranes, which membranes are usually arranged in a stack. Several different specific transfer embodiments are provided. Even though perhaps not explicitly enumerated, all variations and combinations of the described methods are encompassed herein. Wicking Transfer

In particular embodiments, a transfer liquid (such as a buffer) is passed through the membranes to encourage movement of the nucleic acids from the sample to the membranes and through them. A distal or downstream wick may also be provided to help move liquid (such as the buffer) through the membranes in a desired direction of movement.

In general in such embodiments, a membrane stack or array is placed atop a stack of one or more sheets of blotting paper, which acts as a lower wick, pulling buffer out of a buffer chamber(s). This assembly is essentially similar in structure to a conventional Northern (or Southern) blot tower, but for the inclusion of multiple membranes to which the nucleic acids are transferred. A nucleic acid trap (for instance, a nitrocellulose sheet) may be positioned intermediate between the membrane array and blotting paper to help the user ascertain whether and/or to what extent transfer has occurred.

This system can be employed to create "carbon copies" or substantial replicas of the nucleic acid contents of the sample applied to the stack. The membranes are assembled into an array in a layered or stacked configuration. In a particular embodiment, a substantially two-dimensional sample (such as a conventional frozen tissue section or a gel) containing nucleic acids is positioned adjacent to and in contact with one face of the membrane array/stack. Buffer is applied to the gel/membrane assembly, for instance using buffer chambers and wicks, to elute and transfer nucleic acids from the sample and into the membranes. By way of example, the transfer can be carried out for one to several hours, for instance over night. Transfer can be carried out at various temperatures depending on the application; in specific examples, transfer is carried out at ambient temperature. In other embodiments, transfer is carried out at higher or lower temperatures, for instance at temperatures from about 40 °C to about 80 °C, in particular embodiments about 50 °C to about 60 °C, or more particularly in specific examples at 55 °C. In some embodiments, after transfer, the membranes are separated and incubated with the detector molecule (such as hybridization probes). Detector molecules are selected based on the specific target nucleic acids sought. Membranes are washed in a buffer and the nucleic acid / detector complex visualized using one of a number of techniques such as radiography or direct fluorescence. Commercially available flatbed scanners and digital imaging software can be employed to display the images according to the preference of the user.

One specific embodiment is a method for detecting nucleic acids in a tissue section or other sample in which the nucleic acids are arranged in a definable way in relation to each other in at least two-dimensions (e.g, a gel, such as an electrophoretic gel), by creating replicates or "carbon copies" (substantial copies that are not necessarily identical copies, they may have slight differences but can be identical or nearly identical) of the nucleic acids eluted from the starting sample, and visualizing the nucleic acids on the copies using labeled hybridization probes or other molecules having specific affinity for the biomolecules of interest. Thin membranes in a stacked or layered configuration are brought into contact with a face of the sample in the presence of one or more other reagents (for instance, a transfer buffer), and conditions are provided so that the nucleic acids are eluted from the sample onto the membranes of the stack, whereupon the nucleic acids can be visualized using a variety of techniques, such as those set forth herein.

Certain embodiments of the disclosure include a method of detecting a nucleic acid in a biological sample using stacked contiguous layered membranes that peπnit nucleic acids to move through a plurality of the membranes (generally in a direction across the thickness of the membranes and substantially peφendicularly to the face of the membrane stack), while directly capturing at least a portion of the nucleic acids of the sample on one or more of the membranes. Nucleic acids from the sample are moved through the membranes under conditions that allow one or more of the membranes to directly capture the nucleic acids, and nucleic acids of interest are concurrently or subsequently detected on the membranes, for example by exposing the nucleic acids of interest to a detector, such as a specific detector molecule (for example a nucleic acid probe).

Alternatively, the nucleic acids from the starting biological sample itself are detectors (such as a nucleic acid probe) to which a second sample is exposed. In examples of this embodiment, the biological sample comprises one or more purified nucleic acid probes placed in assigned locations on a surface of the stack of membranes (for instance, by being transferred from an array), which probes are allowed to migrate through and into the membranes (for example in a direction of movement across the thickness of the membranes in the stack) to produce multiple substantial "copies" of the original pattern of probes, with a portion of each nucleic acid sample in a location on each of the multiple membranes that substantially correspond to its location in the starting sample. The membranes then can be separated from each other and exposed to a target biological specimen (e.g., a tissue section or a sample provided in a fluid, for instance a sample in a hybridization solution), which may include nucleic acid molecules that hybridize to or otherwise specifically interact with the probes on one or more of the multiple membranes. In some examples, the starting biological sample is a tissue specimen (such as a tissue slice or section) that is placed on a face of the stack of membranes, and nucleic acids from the tissue specimen are captured by the membranes as they move through the membranes. The membranes may, for example, be separated prior to detecting the nucleic acids of interest, and the separated membranes are then exposed to one or more detector molecules. Alternatively, nucleic acids of interest may be contained in a biological specimen (other than a tissue specimen) to which the membranes are exposed.

Nucleic acids detected on the membrane copies may be correlated with a biological characteristic of the sample. For example, a tissue specimen may be placed in a position on a face of the stack for the transfer, and a nucleic acid of interest (such as a particular mRNA) may be detected in one of the membrane copies at a position (in the two-dimensions that characterize the face of the membrane and the face of the sample placed in contact with the membrane stack) that corresponds to the position in which the tissue specimen (or one of its substructures such as an organelle) was placed. The presence of that nucleic acid in the tissue specimen can then be correlated with a biological characteristic of the sample, such as a disease state, developmental state, treatment response, and so forth. For example, a highly malignant tissue specimen may be found to contain a mRNA that may then be associated with the highly malignant phenotype of the specimen.

In particular examples, provided methods can be used to create a set of microarray substantial "copies" by applying a plurality of detectors, such as DNA probes, to the stack of membranes. The stack of membranes provide a plurality of surfaces through which the probes (generally, detector molecules) move (in a direction substantially peφendicular to the face of each membrane surface), and in which a portion of the probes are directly captured by one or more of the membranes. The membrane substrates can be subsequently separated to provide corresponding substrates having a plurality of DNA probes that are in corresponding positions (relative to their starting positions in which they were originally applied) in relation to each other on each of said substrates. Thus, the multiple membranes maintain a substantially coherent relationship between the probes as they move through the substrate. This coherent relationship may or may not be a direct spatial correspondence, but the relative relationship between the nucleic acids will be maintained in such a way that the identity of the nucleic acids on each of the membranes can be known from the relationship in which the nucleic acids were placed on the stack of membranes. Contact Transfer

There is illustrated in FIG. 2A an alternative embodiment of an apparatus 10 for transferring nucleic acids from a substantially two-dimensional sample 11 onto a membrane stack 13, which stack in some embodiments is provided in the form of a kit. Apparatus 10 generally includes a membrane stack 13 upon which a sample 11 (illustrated as a tissue section) may be placed, a pair of filter pads 24 and 26, and a fluid impervious enclosure 28, such as a plastic bag or the like. Optionally, the sample 11 (e.g., a RNA or DNA gel) may be presented on a support 30 (as illustrated in FIG. 2B). More specifically, in a first embodiment, membrane stack 13 comprises one or more membranes 12, for instance up to five membranes, generally constructed as described herein. The membranes 12 in stack 13 should have a high affinity for nucleic acids but have a low capacity for retaining such molecules. This permits the nucleic acids to pass through the membrane stack with only a limited number being trapped on each of the successive layers, and with the nucleic acids maintaining the same orientation with respect to each other as was present in the original sample. Hence, multiple "carbon copies" of the sample (substantial copies that are not necessarily identical copies, they may have slight differences but can be identical or nearly identical) can be generated. In other words, the low capacity allows the creation of multiple replicates as only a limited quantity of the nucleic acids is trapped on each layer.

First and second filter pads 24, 26 are preferably constructed of a blotting paper such as GB004 Blotter Paper available from Schleicher and Schuell. Filter pads 24, 26 are saturated with a transfer buffer, such as TBE (Tris-Borate-EDTA) or SSC (Saline-Sodium Citrate), optionally including a blocking agent (e.g., Denhardt's solution of Salmon sperm DNA) and/or elution buffer (0.5M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, and polyethylene glycol).

Enclosure 28 may comprise any collapsible, fluid impervious material adapted to envelop the sample 11, membrane stack 13, and filter pads 24, 26, which may be kit components. Enclosure 28 is preferably a plastic bag, such as a heat sealable pouch. By way of example, such a bag may be made of a resin, such as a polyester or other resin. In certain embodiments, enclosure 28 is a heat sealable pouch such as those available from Kapak Coφ. (Minneapolis, MN).

In use and operation, the sample 11 is positioned in contact with a face of a membrane stack 13 and both the sample and stack are placed between two filter pads 24, 26, which have been saturated with transfer buffer, to form an assembled contact transfer stack. The assembled contact transfer stack is placed inside fluid impervious enclosure 28, such as a plastic bag. The membranes are pre-wetted in the aforementioned transfer solution.

Fluid impervious enclosure 28 is placed between a pair of substantially flat surfaces 32, at least one of which may serve as a source of heat. By way of example, the pair of substantially flat surfaces 32 can be surfaces of a pair of heating elements such as those provided in gel dryers manufactured by Bio-Rad Laboratories (Hercules, CA). In other embodiments, the pair of flat surfaces 32 may be provided by MJ Research devices, such as the PTC-200 Peltier thermal cycler, which provide a separate heated lid and a thumbwheel to adjust height and pressure of the lid and thereby provide pressure.

In embodiments where heat is applied only from one side of the assembled sample and stack, the heat is preferentially applied from the side of the sample rather than the membrane stack side, such that a heat gradient is created with the heat applied on the sample side. In some embodiments, to cause transfer from the sample 11 to membrane stack 13, the bag and its contents are heated to a temperature of 40 to 95 °C, in some embodiments 50 to 60 °C, or more particularly in some embodiments 55 °C. The bag and its contents are heated for at least about an hour, and in some embodiments about two hours or more. Sufficient pressure is applied throughout the heating process to ensure that there is adequate contact between the sample and the membrane stack to facilitate transfer of nucleic acids to the membrane stack. By way of example, such pressure can be applied using a weight 34 of 7 grams per square centimeter (though more can be used, for instance, up to about four times this much) of the membrane stack, which may optionally be included as a kit component. Springs, clamps, or clips capable of applying pressure may be employed instead of a weight.

To ensure that the binding capacity of the membranes is sufficiently low to prevent trapping of too much of the sample, in some embodiments the thickness of membrane substrate should be less than 30 microns, in some embodiments from 4 to 20 microns, and particular embodiments from 8 to 10 microns. The pore size of the substrate should be from 0.1 to 5.0 microns, in particular embodiments 0.4 microns. Another advantage of using such a thin membrane is that is lessens the phenomenon of lateral diffusion of the sample and specific elements or features within the sample. In generally, the thicker the stack of membranes, the wider will be the diffusion of biomolecules moving through the stack.

The substrate includes a coating on its upper and/or lower surfaces to increase specific binding of the proteins or other targeted biomolecules. The coating in certain embodiments is nitrocellulose, but other materials such as poly-L-lysine may also be employed.

IV. Types of Samples

Any substantially two-dimensional sample material that contains releasable nucleic acids can be used as a source of nucleic acids in the provided transfer processes. By "two-dimensional" it is meant that the material is, or can be formulated so that it is, substantially flat and relatively thin; it is understood that the sample in fact exists in three dimensions. Representative examples of substantially two-dimensional samples include tissue samples such as thin section slices (e.g., archival or frozen tissue samples), tissue arrays, cDNA or other nucleic acid microarrays, 1-D nucleic acid gels (e.g., agarose or polyacrylamide gels), nucleic acid prints on filter paper, and so forth. In general, such two-dimensional samples contain biomolecules in a definable two-dimensional relationship to each other, which is copied in the replication process.

It is further contemplated that the described transfer methods can be used in forensic procedures to detect and study biological material such as bodily fluids; to detect biological (e.g., microbial) contamination of food or other substances; and so forth. In order to provide the sample in a substantially flat and thin format, substances may be suspended in a liquid or gas, then run through and optionally affixed to a filter such as a sheet of filter paper, with the filter then used as the transfer sample. By way of example, a soil sample or fluid sample could be so prepared for transfer. Some substances may be compressed into a substantially flat form, for instance by rollers or another spreading mechanism; by way of example, a food sample (e.g, ground meat) could be so prepared. Generally these samples can be referred to as structurally transformed samples, because their format is altered to render them substantially two-dimensional prior to transfer onto a membrane stack. Embodiments provided herein may be used to identify nucleic acids in any biological sample including bodily fluids (e.g, blood, plasma, serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), fluid obtained from a joint, and so forth. Additionally, a biological sample can be obtained from any organ or tissue (including an autopsy specimen) or may comprise cells.

In embodiments where the nucleic acid is provided in the form of a gel, it is contemplated that at least any of the following types of nucleic acid molecules could be transferred: labeled and unlabeled PCR products, mRNA, tRNA, labeled RNA, plasmids, genomic DNA, labeled genomic DNA, RNA and DNA size ladders. This is meant to be a representative rather than an exhaustive list, and is not meant to be limiting.

V. Membranes

In particular examples, the membranes are sufficiently thin to allow the biomolecules to move through the plurality of membranes (for example 5, 10, 50, 100 or more) in the stack. Such membranes, for example, have a thickness of less than 30 microns. The membranes may be made of a material that does not substantially impede movement of the biomolecules through the membranes, such as polycarbonate, cellulose acetate, or mixtures thereof.

The material of the membranes may maintain a relative relationship of biomolecules as they move through the membranes, so that the same biomolecule (or group of biomolecules) move through the plurality of membranes at corresponding positions. In such examples, this coherence of relative relationships allows the different membranes to be substantial "copies" of one another, much like a "carbon copy" would be. However, like a "carbon copy" there may be differences between the different "copies" on the different membranes.

In particular embodiments, the membranes comprise a material that non-specifically increases the affinity of the membranes to the nucleic acids that are moved through the membranes. For example, the membranes may be dipped in, coated with, or impregnated with nitrocellulose, poly- L-lysine, or mixtures thereof. In certain examples the membranes are not treated with a material that blocks non-specific binding of the biomolecules to the membranes, at least during transfer of the biomolecules through the membranes. However, in other embodiments, one or more of such blocking agents are added to the membranes, where the amount of blocking agent reduces the amount of biomolecules bound without blocking it altogether. In certain examples, blocking agent may be added to the membranes after transfer of the biomolecules through the membranes, but before or during exposure to the detectors. In some embodiments, a membrane stack includes a number of individual membranes, for instance at least 2, at least 5, at least 10, at least 20, at least 50, or even more in some cases. Membranes in the stack are generally constructed as described herein. Examples of the membranes are constructed of a porous substrate coated with a material that increases the affinity of the membrane to the biomolecules being transferred. The substrate may be constructed of polycarbonate or a similar polymeric material (or blend of materials) that maintains sufficient structural integrity despite being made porous and very thin. This polymeric material may include, for example, polyester, polyethylene, terephthalate, or a cellulose derivative, such as cellulose acetate, as well as polyolefins (e.g., polyethelyle, polypropylene, etc.), gels or other porous materials or mixtures thereof.

Representative membranes have a high affinity for nucleic acids but have a low capacity for retaining such molecules. This binding profile permits nucleic acids to pass through the membrane stack with only a limited number being trapped on each successive layer, thereby allowing multiple "carbon copies" of the nucleic acids in the sample to be generated. In other words, the low capacity allows the creation of multiple replicates as only a limited quantity of the biomolecules is trapped on each layer.

With reference to FIG. 3, individual membranes 12 are constructed of a porous substrate 90 coated with a material that increases the affinity of the membrane to the nucleic acids being transferred. Substrate 90 is, for example, constructed of polycarbonate or a similar polymeric material that maintains sufficient structural integrity despite being made porous and very thin. However, in lieu of polycarbonate the substrate 90 may for example be constructed of cellulose derivatives such as cellulose acetate, as well as polyolefins, (e.g, polyethylene, polypropylene, etc.).

The illustrated membrane 12 includes a coating 92 on its upper and lower surfaces to increase non-specific binding of the nucleic acids. Although the binding of nucleic acids to the coating is "non-specific" in the sense that it does not recognize particular nucleic acids, it may be specific in that it recognizes and specifically binds classes of biomolecules (such as nucleic acids) and not substantially to others, such as proteins.

Coating 92 in specific disclosed embodiments is nitrocellulose, but other materials such as poly-L-lysine may also be employed. Before being applied to substrate 90, the nitrocellulose (or other coating material) is dissolved in methanol or other appropriate solvent in concentration from 0.1%-1.0%, though in some embodiments the concentration can be lower or higher. The membranes are immersed in this solution. In lieu of coating 92, nitrocellulose or other materials with an affinity for nucleic acids can be mixed with the polycarbonate or other porous substrate material before the membrane substrate is formed, thereby providing an uncoated substrate having the desired characteristics of the membrane. Alternative coating methods known in the art may be used in lieu of dip coating, including for instance lamination or spray coating. Alternatively, only one surface may be coated, such as the surface that faces the sample, instead of both surfaces. With reference to FIG. A, the aforementioned technique may be described as "direct capture" since the target biomolecules 100 are captured directly on a surface of membranes (or within the membrane), instead of being captured indirectly by a binding agent (such as a nucleic acid probe) that itself is applied to the membrane. During this disclosed process, different components of the sample bind to the membrane with the same affinity, but directly proportional to their concentration in the sample (a component with a higher concentration will leave more molecules on each membrane, and a component with a lower concentration will leave fewer molecules on each membrane). A detector molecule 104, such as a labeled hybridization probe that specifically binds to the biomolecule 100 at illustrated sequences 102, may be utilized to detect nucleic acids bound to the membrane. In examples in which the amount of a component bound to the membrane is proportional to the amount of the component in the sample, an amount of the detector molecule can be correlated to an amount (or relative amount) of the biomolecule detected.

In order to achieve high affinity and high capacity for a particular group of biomolecules from a sample, coating of the membranes with a captor molecule 106 is performed as described herein. This method is analogous to the method of providing membranes specific for capturing specific proteins, as described by Englert et al. (Cancer Research 60:1526-1530, 2000). This may be referred to as "indirect capture" and is illustrated in FIG. 5. Captor 106 can be cDNA or another nucleic acid molecule preparation. Single-stranded cDNA molecules generated by a number of means (such as polymerase chain reaction or another type of in vitro amplification procedure, nick translation, reverse transcription, oligonucleotide synthesis) can be directly attached to the membrane. Alternatively, linker-arms that would allow spatial control of the captor binding could be directly attached to the membrane followed by captor molecule attachment to the linker arms. This will expose the majority of the active target recognition sites increasing that way capacity of the indirect capture. By way of example, streptavidin coated membranes may be employed to bind end- biotinylated nucleic acids.

In another embodiment, each of the membranes comprise a ligand coating (e.g., a unique ligand coating, in that it is different from the others in the stack, or different from at least one other in the stack) that selectively binds to a nucleic acid species in the biological sample based on a particular sequence of the nucleic acid. As a result, the membranes function to fractionate the nucleic acids in the sample rather than replicate them as with membranes in other described embodiments.

The coating could be made in many different ways so that each membrane binds a selective subset of the total nucleic acid content in the sample. Other Membrane Characteristics

Membranes in some embodiments are constructed in the manner disclosed in PCT International Application PCT US01/44009, filed on November 20, 2001 (which is incoφorated herein by reference in its entirety). In some embodiments membranes are constructed of a thin porous substrate that may be coated with a material to increase the affinity of the membrane to nucleic acids being transferred thereto.

It is a particular feature of some embodiments that membranes used for the transfer have a high affinity for nucleic acids, but have a low capacity for retaining such molecules. This feature permits the nucleic acids to pass through the membrane stack with only a limited number being trapped on each of the successive layers, thereby allowing multiple replicate "carbon copies" to be generated. In other words, the low capacity of the membrane material allows creation of multiple replicates, since only a limited quantity of the nucleic acids are trapped on each layer. More specifically, in specific embodiments the affinity and capacity of membrane should be such that when at least three, and preferably ten or more, membranes are stacked and applied to (or placed in contact with) a sample according to one of the provided methods, most of the nucleic acids of interest can be detected on any and all of the membranes, including those positioned furthest from the sample. If a membrane were used that had a high binding capacity- such as the transfer membranes used with conventional gel blotting, multiple replicas could not be made in this manner unless the binding capacity of the membrane was overwhelmed by the amount of biomolecule applied to the membrane.

To maintain the binding capacity of membrane sufficiently low to avoid trapping of too much of the sample, the thickness of the substrate is, for example, less than about 30 microns, and in particular embodiments is between about 4-20 microns, for example between about 8 to 10 microns. The pore size of the substrate is, for example, between about 0.1 to 5.0 microns, such as about 0.4-0.6 microns, and more specifically 0.4 microns. Another advantage of using a thin membrane is that is lessens the phenomenon of lateral diffusion. The thicker the overall stack, the wider the lateral diffusion of biomolecules moving through the stack. Alternatively, a thicker membrane could be employed with binding sites blocked so as to lower its binding affinity for nucleic acids.

It will be appreciated that because the size of the membranes in the stack/array can be varied, the user has the option of analyzing a large number of different samples in parallel, thereby permitting direct comparison between different samples, such as patient samples (e.g., different patient samples, or patient samples and a reference standard, or samples of different tissues or species, etc.). For example, different samples from the same patient at different stages of disease can be compared in a side-by-side arrangement, as can samples from different patients with the same disease.

In some embodiments, the membrane substrates are "track-etched membranes" (a/k/a "screen membranes"); a representative track-etched membrane is shown in Figure 15. Track-etched membranes are formed, for example, by exposing a dense film to ionizing radiation to form damage tracks, which are then etched by a strong alkaline solution. This process creates well-defined pores. Further details of this process may be found on the Internet site of Osmonics (Minnetonka, Minnesota) under the heading "Basic Principles of Microfiltration." In other embodiments, it may be desirable to increase the surface area of track-etched membranes, which have a very smooth, flat surface, by coating the surface with, e.g., a fiber or other material to give it a rougher texture. Alternatively the surface area could be scratched or rubbed with an abrasive to increase the surface area. In yet other embodiments, a "depth" or "tortuous pore membrane" (an example of which is shown in Figure 16) may be employed if its capacity is rendered low enough to permit a stack of three or more such membranes to be used according the methods disclosed herein. This could be accomplished, for example, by casting the membrane very thin, far thinner that the thickness of depth membranes conventionally employed (i.e., approximately 150 μm). Alternatively, blocking certain binding sites could lower the capacity (e. g. , the capacity to bind nucleic acids) of conventional depth membranes. For instance, the membranes could be pretreated with a nucleic acid preparation, such as tRNA or salmon sperm DNA, to block some of the available binding sites on the membrane. By way of example, relatively low concentrations (compared to the level used for blocking a membrane) of non-specific nucleic acid could be used to pretreat the membrane. The exact level of nucleic acid used for pretreatment can be readily determined for each application by titrating the concentration for treatment of a number of membranes, then comparing the relative ability of the pretreated membranes to capture specific nucleic acid transferred thereto using methods described herein. By way of example, membranes can be partially blocked using solutions of about 0.001 to about 1 μg/μl of a blocking molecule preparation, such as tRNA or salmon sperm DNA. Some non-limiting examples of membrane substrates that may be employed in methods provided herein include the Isopore™ (polycarbonate film) membrane available from Millipore (Bedford, Massachusetts), the Poretics® polycarbonate or polyester membranes available from Osmonics (Minnetonka, Minnesota), and the Cyclopore™ polycarbonate or polyester membranes available from Whatman (Clifton, New Jersey). Framed membranes

In another embodiment, with reference to FIGS. 6-8, framed membrane stack 110 comprises a plurality of individual membrane units 112 releasably secured to one another. Each membrane unit 112 comprises a membrane 12 having a frame 114 mounted about the periphery thereof. Membrane unit 112 can vary in size but should be large enough so that membrane 12 can overlay a typical electrophoresis gel or other sample.

The number of membrane units 112 included in stack 110 can vary depending on the number of nucleic acids to be detected from the gel. For most applications, from 3 to 25 or more membranes will be sufficient, for instance from 5 to 15, or from about 10 to 12. The entire thickness, Ts, of stack 110 (FIG. 6) is in some embodiments no more than about 0.25 cm. In some embodiments, in order to give each membrane sufficient rigidity to enable it to be separated the other membranes in stack 110 and individually processed, a frame 114 is mounted onto the periphery of membrane 12 thereby forming membrane unit 112. Frames 114 preferably comprise a generally "U" shaped configuration covering three sides of the membranes while defining an open space or gap 120 that functions as a channel to permit the manual removal of air pockets or fluids in the manner described.

The composition and dimensions of frame 114 should be such that the frame provides sufficient rigidity for the user to grip the frame with one hand and manipulate the membranes as needed. At the same time, the frames must be sufficiently thin so that when stacked they do not interfere with nucleic acid transfer from the gel onto the membrane stack 110. Each membrane 12 in stack 110 should be capable of making direct contact with adjacent membranes during the transfer processes described herein.

The width W (FIG. 8) of frame 114 is preferably between about 0.3 to 0.7 cm and the thickness of the frame, Tf, is between about 0.005 to 0.03 cm, most preferably about 0.01 cm thick. Thus, frame 114 is about ten times thicker than membrane 12. In certain embodiments, the materials that comprise frames 114 are able to maintain their structure and function at temperatures of up to 80° C but are able to melt when applied to a typical heat-sealing apparatus. One skilled in the relevant art will readily appreciate that a variety of compositions and configurations of frames 114 could meet these requirements. Examples of materials that may be employed to make frames 114 are transparency film available from Canon or any thin plastic sheet made of polycarbonate, polyester, polyvinylchoride, or polyvinilechloride.

As viewed in FIG. 7, a pair of outwardly depending tabs 116 is defined by frame 114. Each tab is adapted to be sealed to the corresponding tab on an adjacent membrane so as to hold stack 110 together during the gel transfer process. After the nucleic acids are transferred onto the membranes tabs 116 are cut with a scissors so that the membranes may be separated and incubated in separate detection solutions.

At least one side of frame 114 defines a surface 118 upon which indicia may be imprinted. The indicia may include the name of the product or manufacturer, and/or the membrane number. Machine-readable indicia such as a bar code or the like (not shown) also may be provided.

Frames 114 may be mounted to the perimeter of membranes 12 by various means readily familiar to those skilled in the art, including use of adhesives such as rubber cement or 3M adhesive or conventional heat-sealing or laminating techniques.

VI. Analysis of Membrane Replicates

After transfer, the processed membranes (or layers) can be separated and each incubated with one or more different detector molecules (such as nucleic acid hybridization probes) specific for particular target nucleic acids of interest. In certain embodiments, the detectors/probes employed are labeled or otherwise detectable using any of a variety of techniques, such as chemiluminescence. Thus, while in some embodiments each membrane has essentially the same pattern of biomolecules bound to it, different combinations of biomolecules can be made observable on each membrane by selecting particular probes to be applied and detected. In some embodiments, there is no need to incubate the separated membranes with a detector molecule. For instance, in some specific methods one or more of the molecules that is transferred to the membrane stack is detectable, e.g., by being labeled with a marker moiety.

Digital images of membranes may be created using a variety of instruments including the Image Station® CCD instrument available from Kodak Scientific Imaging (New Haven, CT). Alternatively, images may be captured on film (such as X-ray film) and digitalized by flat bed scanners. Software can be employed to align the images and perform densitometry functions. The user can select the region of interest for analysis and the signal intensities are recorded and normalized. The numerical intensity values are then compared. In many embodiments, the detectors/ligands employed are labeled or otherwise made detectable using any of several techniques, such as enhanced chemiluminescence (ECL), fluorescence, counter-ligand staining, radioactivity, paramagnetism, enzymatic activity, differential staining, nucleic acid amplification, etc. The membrane blots are preferably scanned, and more preferably digitally imaged, to permit their storage, transmission, and reference. Such scanning and/or digitahzation may be accomplished using any of several commercially available scientific imaging instruments (see, e.g., Patton et al, Electrophoresis 14:650-658, 1993; Tietz et al, Electrophoresis 12:46-54, 1991; Spragg et al, Anal Biochem. 129:255-268, 1983; Garrison et al., J Biol. Chem. 257:13144-13149, 1982; all herein incoφorated by reference).

VII. Kits

Other embodiments of the disclosure include kits for use with direct capture embodiments, which kits contain a membrane array or stack for detecting nucleic acids in a sample. The array includes a plurality of membranes, each of which has a non-specific or substantially same affinity for the biomolecules. Still other kit embodiments are provided for use with indirect capture embodiments, wherein the provided membrane array or stack includes two or more individual membranes with different affinities for different target nucleic acid molecules.

Certain provided kits also include one or more containers of detector molecules, such as labeled hybridization probes (or mixtures of probes), for detecting nucleic acids captured on at least one of the membranes. In particular examples of the kit, the membranes are polymer substrates containing or coated with a material (such as nitrocellulose) for increasing an affinity of the substrate to the nucleic acids.

Kits may additionally contain reagents for effecting the detection of detector/ligand-nucleic acid binding, buffer, and/or instructions or labels that indicate the particular detector or detector cocktail to be applied to a particular membrane. Software, such as that discussed herein, may also be included in the kit or may be accessible via modem, the Internet, by mail, or by other means. The methods and kits allow up to several thousand discrete nucleic acids to be identified, annotated, and, at the user's option, compared to the pattern of nucleic acids generated from other biological samples stored in a database.

Also provided is another specific embodiment, directed to a method and a kit for identifying (i.e. detecting, annotating, and/or characterizing) groups of nucleic acids that have been separated by gel electrophoresis. Examples of such kits comprise at least one of the following components: (i) a membrane stack or framed membrane stack (as illustrated) upon which the nucleic acids are transferred, (ii) nucleic acid transfer reagent(s) and (iii) nucleic acids detector molecules, such as double-stranded DNA-specific chelators (e.g., certain dyes) or sequence-specific detector molecules (e.g., hybridization probe molecules). The kit may also include software that allows the user to analyze and manipulate the images produced so as to compare them to images from other samples in a database. Alternatively the software may be acquired or accessed independent of the kit.

In some embodiments, transfer reagent is also provided with a kit. Examples of transfer reagents include SSC, TBE, methanol, and so forth. Specific examples of transfer reagent suitable for use in examples of such kits are in the Examples.

In addition to identifying nucleic acids of interest based on sequence, other kits are provided that can be employed with the disclosed methods of nucleic acid sequence analysis, pre-transfer nucleic acid amplification, and so forth.

IX. Applications

At present, a Northern blot is performed to analyze the amount of an individual mRNA species that is present in an RNA population. The RNA is separated on an agarose gel, and then blotted to a nitrocellulose membrane. A gene-specific labeled probe is hybridized to the blot and used to identify the size and relative amount of the mRNA species of interest. Only one gene is analyzed per experiment.

The LES-based Northern blot methods provided herein significantly increase the number of mRNA species that are analyzed in each experiment.

Also provided herein are methods of specifically hybridizing mRNA species to loaded membranes, using indirect capture of the target molecules. In one example of such a method, an RNA sample is separated by electrophoresis on a standard agarose gel. The entire mRNA population is then radioactively labeled in the gel in-situ using standard oligodT-mediated reverse transcription. The labeled fragments are then transferred through a stack of LES membranes per any of the exemplified protocols, and the two dimensional relationship of the nucleic acid bands in the gel is maintained. Each LES membrane is coated with DNA specific for an individual gene. As the nucleic acids traverse the LES membranes, the pieces of labeled DNA corresponding to a specific mRNA species in the gel bind specifically to the LES membrane that is coated with a capture DNA for that sequence, i.e., each LES membrane "pulls out" the labeled fragments of its corresponding sequence. Thus, each membrane becomes a "Northern blot" of an individual gene. Because tens (even hundreds) of LES membranes can be used per transfer, each mRNA sample can be simultaneously analyzed for multiple different genes.

A variation on the method described above can be utilized to further increase the number of transcripts that are analyzed in each LES-Northern blot. In this embodiment, each LES membrane is coated with DNA corresponding to two or more separate genes, for instance ten genes. The sequences used to coat the membranes are selected such that the corresponding mRNA transcripts are all of different sizes, i.e., transcript #1 is one kb in length, transcript #2 is two kb in length, etc. In- gel labeling of the entire mRNA population and subsequent LES transfer are performed as described above. In this embodiment, each LES membrane "pulls out" labeled cDNA corresponding to ten different genes, each of which can be discriminated on the LES membrane due to its specific size.

Thus, this version of the LES-Northern method permits simultaneous analysis of a very large number of mRNA species since one could use tens (even hundreds) of LES membranes, each membrane capable of analyzing the expression level often genes.

Another variation of the transfer method is used to increase the sensitivity of the technique, permitting multiple analyses of small amounts of nucleic acid starting material (e.g., mRNA).

Examples of this embodiment provide a nucleic acid amplification step in the method. For example, mRNA can be run on an agarose gel by known methods. Then, all of the transcripts in the gel can be subjected to in-gel-RT-PCR such that each mRNA/cDNA is amplified significantly, and optionally labeled. The optionally labeled amplified products in the gel are then transferred into a set of membranes as described herein. Thus a highly sensitive, multiplex Northern blot is produced.

In another embodiment, cellular nucleic acids (e.g., genomic DNA or mRNA) present in tissue sections or other cell-based samples are amplified in situ, and the amplified products transferred onto membranes by methods described herein. For instance, in addition to tissue sections, this approach can be applied to cytologic smears, or other samples consisting exclusively of loose cells, for example pap smears or cell aspirates. In such situations, amplification and generation of multiple copies of the sample is particularly valuable, since the original sample is always unique, i.e. no "recuts" can be generated as they could from a block of archival material. By generating copies of these samples, it would be possible to obtain multiple analyses from a single sample.

In yet another embodiment, nucleic acids in a sample (e.g., a tissue section or gel) are transferred from the sample into a plate or tray or the like containing multiple wells, using a physical transfer method such as centrifugation (for instance, using methods such as those described herein or in U.S. patent application number 60/428,754 (Methods and Apparatus for Performing Multiple Simultaneous Manipulations of Biomolecules in a Two-Dimensional Array), filed November 25, 2002, incoφorated herein by reference) or microdissection. The nucleic acids then can be amplified in the multi-well plate prior to transfer to membranes as described herein.

Embodiments employing indirect capture transfer technique present several advantages to conventional nucleic acid blotting techniques. By transferring labeled nucleic acids, they automatically serve as the "probe," and no additional hybridization steps are necessary after the transfer. This could be applied for several puφoses, including: a) multiple sequencing reactions electrophoresed together on a gel, and then sorted out by gene-specific coated membranes; b) Northern blot analysis transferred through gene-specific membranes, and then hybridized with a second probe, increasing the specificity of the detection, since a two step hybridization is applied to detect the signals (analogous to a sandwich immunohistochemical reaction for protein analysis); and c) total RNA RT-PCR analysis of one sample, then sorting out the genes of interest by transfer the entire preparation of cDNAs through gene-specific coated membranes.

Similar to the embodiments described for generating replicate Northern (RNA) blots, consistent replicates can also be obtained for Southern (DNA) blotting. Southern blotting analysis is applied to identify molecular alterations that have important diagnostic and prognostic clinical significance, such as amplification or re-arrangement analysis (Poremba et al, Clin. Padiatr. 213: 186-190, 2001; O'Sullivan et al, Hum Pathol. 32: 1109-15, 2001; Tomescu & Barr, Trends Mol Med 1: 554-9, 2001; Anderson et al, Br J Cancer 85: 831-5, 2001; Bergsagel & Kuehl, Oncogene 20: 5611-22, 2001; Kuppers & Dalla-Favera, Oncogene 20: 5580-94, 2001; Crans & Sakamoto, Leukemia 15: 313-31, 2001). As an example, identification of specific translocations or amplifications are important diagnostic tools in poorly differentiated tumors such as small blue round cell tumors, including neuroblastomas (Poremba et al, Clin. Padiatr. 213: 186-190, 2001), rhabdomyosarcomas (Tomescu & Barr, Trends Mol Med 7: 554-9, 2001 ; Anderson et al. , Br J Cancer 85: 831-5, 2001), and Ewing/peripheral neuroectodermal tumors (O'Sullivan et al, Hum Pathol. 32: 1109-15, 2001). Several translocations have been identified on hematologic malignancies such as B cell lymphomas (Kuppers & Dalla-Favera, Oncogene 20: 5580-94, 2001), myelomas (Bergsagel & Kuehl, Oncogene 20: 5611-22, 2001) and leukemias (Crans & Sakamoto, Leukemia 15: 313-31, 2001), many of them playing a key role in the biology of the tumors. Some solid tumors also present characteristic translocations (Pang et al. Genes Chromo. Cancer 33: 150-9, 2002). By using a Southern replicate approach, several probes can be analyzed in a single experiment on the samples. Nucleic Acid Sequence Analysis

Yet a further application of the transfer methods is to perform multiplex analysis of DNA sequencing gels. As an example, simultaneous analysis of multiple sequencing reactions is performed as follows: An investigator runs, for instance, sequencing reactions on ten different DNA fragments of interest. Rather than loading the products of the sequencing reaction into separate lanes of a sequencing gel (i.e., a separate lane for the products of each DNA species that was sequenced), all of the products are loaded together into one lane. The DNA in the gel is then transferred through ten membranes, each of which is coated with DNA corresponding to one of the ten DNA fragments that were sequenced. Each LES membrane hybridizes with (i.e., pulls out) the corresponding "ladder" of sequencing products that are specific for that DNA fragment. Thus, by analyzing each membrane, the sequence of all of the DNA fragments can be determined.

This embodiment can increase the yield of sequencing gels by 10-fold (or more, depending on the number of membranes that are utilized in each transfer). This embodiment can also be used to perform direct sequencing from complex DNA templates, such as whole chromosomes or genomes. Using previously known procedures, it is not possible to perform direct sequencing from a complex DNA mixture. This is because the sequencing primer will anneal to its intended target sequence, but will also anneal to multiple similar non-target sequences that are present in the complex DNA template. Thus, if one performs direct sequencing on a complex DNA sample, a large number of sequencing products are produced in the reaction due to primer cross-reaction with non-target region(s), and the resultant sequencing ladder is not inteφretable. Therefore, it is necessary to perform multiple pre-sequencing steps to prepare small, purified DNA templates that are amenable to sequencing. Depending on the approach that one takes, this may include screening a library to identify/grow/purify a bacterial clone for sequencing, or, performing initial amplification on the complex DNA mixture, followed by purification of the DNA on a gel. In either event, these pre-sequencing steps are laborious, expensive, and time consuming.

Applying the herein described method solves problems associated with direct sequencing of complex DNA template. Moreover, the method is also believed to permit multiple, simultaneous sequencing reactions to be performed on a genomic DNA and analyzed. This can be accomplished as follows: A whole-genome sample is subjected to a simultaneous sequencing reaction using ten primers against ten different genes. During the sequencing reaction, the ten primers anneal to their intended sites in the genome, as well as to multiple, non-specific sites. Thus, the products of the reaction include ladders representing the sequence of the ten genes, as well as sequence ladders that were derived from non-specific hybridization of the primers. The entire reaction is then run in a single lane on a gel. The gel is then transferred through a stack of LES membranes, each of which is coated with DNA representing one of the ten genes of interest. All of the non-specific sequencing products pass through the LES membranes without hybridizing to them. However, the ladder of gene-specific sequencing products hybridizes to each corresponding LES membrane, thus allowing the sequence of each DNA fragment to be determined. Nucleic acid amplification

As discussed above, the amount of nucleic acid (for instance, mRNA) available from a sample may be too low to reliably transfer to LES membranes. For instance, it is generally not possible to produce 20 μg of polyA RNA from certain biopsy samples, microdissected samples, aspirates, environmental samples, and so forth. In these instances, it is beneficial to amplify nucleic acids in the sample.

Amplification can be carried out at any point in the method, e.g., before separating the sample on a gel or after the sample has been electrophoresed ("in gel-o" amplification). In situ amplification (e.g., in a tissue section or laser microdissected sample) can similarly be used to increase the amount of nucleic acid prior to transferring a sample into and through a membrane stack. In addition, it is contemplated that "in stack-o" and "in membran-o" amplification can be used to amplify nucleic acids after they are transferred from a sample, using techniques based on those used for in situ amplification. X. Image Analysis Software

Software is made available to users of any of the provided kits by providing it on a diskette to be included with the kit, or by making it accessible for downloading over the Internet or a private Intranet network, or by other means. The function of such software is to translate the visible signals generated by detector molecules (such as labeled hybridization probes) into useful information about the nucleic acids of the sample being tested. This information includes the quantity of the nucleic acid(s) in the test sample relative to a control. Suitable software can be obtained from, or adapted from, any of a variety of sources. Image analysis starts with digitalized image(s) of the experimental membranes. As the first step, the user matches templates with the membranes. The software then compares an image of the template and an image of the membrane and performs alignment of spots/bands on the membranes or different membranes. The user has options of visual alignment control and the ability to hand correct minor discrepancies. The second step of analysis will include densitometric readings of the spots on experimental membranes. This data is stored in the database. The third step includes numerical data manipulation. Intensity values of each experimental spot on the membrane are divided with values of the landmark spots. This step generates normalized intensity values for each spot on the membrane. All the spots/bands of interest can thus be compared with each other.

Software preferably allows the user to select the kind of comparative analysis to be performed (i.e., comparing the spots or bands present in one sample with those in another sample or comparing those present on one membrane with those of another membrane within the same membrane stack). Results of the analysis are displayed in, for instance, tabular format and the user is given the option to go back and compare magnified sections of the images of interest.

Having now generally described the invention, embodiments of the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting in any way.

EXAMPLES Example 1: Northern Blot Replicates

This example provides one embodiment of an ultrasensitive nucleic acid blotting technique based on LES, which allows generation of many replicate blots from one sample for RNA (Northern) analysis, significantly increasing the output of the analysis. Methods and Materials Generation of Replicates for Northern Blotting

T-T membranes (20/20 GeneSystems, Rockville MD) were pre-treated treated in 10 x TBE for 30 minutes, washed in 20 x SSC for 5 minutes, then placed on top of each other (stacked), and the stack placed on top of the gel for the transfer.

Fifteen to 20 μg aliquots of cell line RNAs (MDA453, He-La, Osteosarcoma MG-63) and commercially produced cDNA (actin and GPDH from Clontech, Palo Alto, CA; EGFR, cyclinD, cdc2, PCNA, c-myc, HPV-18 from Maxim Biotech, San Francisco, CA) were electrophoresed in a 1%, denaturing agarose gel. After washing the gel twice for five minutes each in DEPC water, a capillary transfer was performed, overnight using the following setup (from bottom to top): 20 x SSC transfer buffer, transfer paper (Gel Blot Paper, Schleicher & Schuell, Keene, NH), agarose gel, 10 T- T (#1-10) membranes (20/20 GeneSystems, Rockville MD), a nitrocellulose trap, 20-40 layers of transfer paper, and a standardized weight (7 g / cm²).

For comparison, a parallel transfer tower was assembled and transferred under the same conditions, replacing the stack of T-T membranes with a conventional nitrocellulose membrane (Prototran, Schleicher & Schuell, Keene, NH).

After transfer, the membranes were then UV-crosslinked (1,200 mJ). Total RNA capture by the membranes was assessed by SYB DX DNA Blot Stain (Molecular Probes, Eugene, OR). Quantitation of the signals was performed using ImageQuant software (IQMac vl.2). Stripping of T-T membranes was performed in boiling 0.5% SDS solution, for 5-10 minutes and membranes were re-blotted. Specific Hybridization to Membranes

After the transfer, individual membranes were pre-hybridized in 6 x SSC, 0.5% SDS, 10 μg/ml salmon sperm DNA, 5 x Denhardt's, at 55 °C for 30 minutes. Twenty five to 50 ng of each probe were random-labeled using the Rediprime II kit (Amersham Pharmacia Biotech,

Buckinghamshire, England) and ³³P incoφoration. The membranes were hybridized using 2,000- 10,000 cpm/μl of labeled probe, at 55 °C, overnight, then washed 2 x 10 minutes in SSC 0.5% SDS, and 2 x 10 minutes in 1 x SSC, 0.1%) SDS. The hybridized blots were exposed using a Phosphorimager 445 SI (Microdinamic Engineering, Rockville MD). Quantitation of the signals was performed using ImageQuant software (IQMac vl .2).

Results In order to generate replicates for Northern blotting, 10 μg of total RNA was electrophoresed on a denaturing gel and 10 pre-treated LES membranes were stacked for capillary transfer out of the gel. The membranes were UV-crosslinked, then a) total RNA was stained (Sybr Dx Blot Stain, Mol Probes) to quantitate transfer on each layer, and b) hybridization was performed using different probes, including housekeeping genes such as beta actin (2.1 kb) and GAPDH (1.2 kb) to assess intensity ratio of the nucleic acids in each layer. Figure 9 shows the total RNA staining of 10 T-T membranes after transferring 15 μg of MDA453 cell line RNA. A typical RNA pattern with both 28S and 18S bands and a smear is seen in all the membranes. The quantitation results of the smears are shown in Table 1. The signal of each of the 10 T-T membranes is expressed as the percentage of the sum of the signals in the 10 membranes. A small variation of intensity of signals is seen between the membranes, from 8-13%. Occasionally a decrease to 4-5% of capture was seen on membranes 9 and 10.

Table 1. Quantitation of total RNA staining in 10 T-T membranes

Figure 10 shows the hybridization of GAPDH on membranes 2, A, 7, and 8 after transferring 15 μg of total RNA from Osteosarcoma MG-63 cell line. A specific 1.3 kb band is seen in all the membranes. The quantitation of the GAPDH signal on 10 T-T membranes is shown in Table 2, again expressed as a percentage of signal on each membrane.

Table 2. Quantitation of GAPDH signals in 10 T-T membranes

Figure 11 shows a comparison of GAPDH signal between a T-T membrane and a conventional nitrocellulose membrane, run in parallel. Fifteen μg of total Osteosarcoma MG-63 cell line RNA were transferred in 2 parallel experiments, one using 10 T-T membranes, another one nitrocellulose membrane. The membranes were processed and exposed in parallel. The quantitation shows that the bands on the T-T and nitrocellulose membranes correspond to 45% and 55% of the total signal, respectively.

Discussion Transfer of RNA into 10 T-T membranes yielded a highly homogeneous distribution of the signals through-out the membranes, assessed both by total RNA staining (Fig. 9) and hybridization of specific probes (Fig. 10). Only occasionally layers 9 and 10 showed a slight decrease in the capture of material to about 4-5% of the total, especially if less than 15 μg of total RNA was used in the original sample. However, after a longer exposure of 48-72 hours, even in these cases a consistent signal was present in these membranes.

When comparing the intensity of signals between one T-T layer of a 10 layer transfer experiment and the nitrocellulose signal on an identical experiment of a conventional Northern blotting, the proportion of the signals were comparable (45% of signal on the T-T layer, and 55% on the nitrocellulose, Fig. 11). This is interesting, since it would be reasonable to expect that the distribution of the original signal on a 10-layer system would result on each layer capturing about

10% of the original signal (assuming a homogeneous distribution of the signals). However, each T-T membrane results in a signal that is comparable to the one obtained in a regular Northern blot transfer. This demonstrates that, on conventional nitrocellulose membranes, not all of the RNA that is transferred translates into a stronger signal. Therefore, the Northern blotting analysis using the T-T membrane system is an ultrasensitive technique.

The STD and variance of the signals in each T-T layer is comparable to those seen on regular nitrocellulose. This shows that multiple Northern analysis based on LES is a reliable, consistent method for studying several samples in one experiment. Different probes can be tested, each one against a different layer, and normalization of the experiments can be performed by comparing the signals to one layer where an internal control such as actin or GAPDH is used. This approach allows for a high throughput Northern-blotting analysis of several genes in a single experiment, on the same samples. This is relevant not only for saving time and effort, but it is applicable in cases where the validation is performed on precious samples, such as human tissue specimens, and the yield of RNA is limited.

Example 2: Hybridization Capture Method

Transferred nucleic acids can be directly analyzed on the membranes by allowing them to hybridize on gene-specific coated layers. This example describes an indirect transfer embodiment, in which target nucleic acids are captured on individual membrane layers by transferring labeled RT- PCR products through gene-specific, coated membranes.

To examine the specificity of the binding, several experiments using different combinations of blank membranes (pre-treated in 10 x TBE), blocked membranes, and gene-specific coated membranes were performed to assess the specificity of the binding. Methods and Materials Preparation ofradiolabeled samples cDNA was synthesized using 1 μg of total RNA as previously described (Chuaqui et al, Urology 50: 301-307, 1997), and PCR incoφorating ³³P for different genes was performed, including beta microglobulin, actin, HPRT (using the primers listed below). The labeled products were electrophoresed on a 1% agarose gel, and then transferred into the membranes essentially as described in Example 1. Preparation of membranes To block the membranes, after the TBE pre-treatment, they were incubated in hybridization solution containing 10 μg/μl salmon sperm DNA and then UV-crosslinked.

To coat the membranes with a specific capture molecule, plasmid clones or non-labeled PCR products for different genes were used, including: beta microglobulin (370 bp) amplified with the β 2-M-5' and β 2-M-3 'primers (SEQ ID NOs: 1 and 2); beta actin (479 bp) amplified with the β act-5' and β act-3 ' primers (SEQ ID NOs: 3 and 4); HPRT (469 bp) amplified with the HPRT-5 ' and

HPRT-3' primers (SEQ ID NOs: 5 and 6); c-myc (479 bp) amplified with the c-myc-5' and c-myc-3' primers (SEQ ID NOs: 7 and 8); c-fos (612 bp) amplified with the c-fos-5' and c-fos-3' primers (SEQ ID NOs: 9 and 10); and c-jun (409 bp) amplified with the c-jun-5' and c-jun-3' primers (SEQ ID NOs: 11 and 12). One to 20 μls of non-labeled, gene-specific PCR products or plasmids was placed on the center of individual membranes, allowed to wick onto the surface of the membrane, air-dried, and UV cross-linked. All the membranes were then pre-hybridized in 6 x SSC, 0.5% SDS, 10 μg/ml salmon sperm DNA, 5 x Denhardt's, at 55 °C for 30 minutes. Transfer and analysis The pre-hybridized (loaded) membranes were then stacked and the transfer performed from the gel as described for the generation of replicates (Example 1), except that it was performed at 55 °C, and only one or two transfer papers were used. The transfer system was incubated at 55 °C overnight, to allow the hybridization to take place. The membranes were then washed and exposed as described as above.

Results and Discussion LES layers were coated with non-labeled PCR products or clones as capture molecules. ³³P labeled RT-PCR products or ³³P end-labeled mRNA molecules were electrophoresed on an agarose gel and transferred through a combination of blank, blocked, and gene-specific coated T-T membrane layers, to assess the effectiveness of specific hybridization and indirect capture of target nucleic acids on the layers during the transfer.

Figure 12A shows the results after transferring a beta microglobulin 370 bp product through a combination membranes, including blank, blocked, beta microglobulin-coated and actin-coated membranes. A specific capture is seen on the beta microglobulin-coated membranes. The signal is also seen on the last blank membrane, which served as a trap to assess the movement of the probe.

Figure 12B shows the results after loading c-myc + c-jun RT-PCR products on lane 1, c-jun on lane 2, and c-myc on lane 3 of an acrylamide gel. The products were then transferred through T-T membranes, one coated with c-myc + c-jun RT-PCR products, 2 with c-jun, and 3 with c-myc. A specific hybridization signal is seen on the corresponding membranes.

Specific capture of the nucleic acids on the LES membranes was achieved. This example demonstrates that, by coating individual membrane layers with a specific template capture molecule, target nucleic acids hybridize specifically to the corresponding layer while moving through the stack.

Example 3: Southern Blot Replicates

This example provides one embodiment of an ultrasensitive nucleic acid blotting technique based on LES, which allows generation of many replicate blots from one sample for DNA (Southern) analysis and significantly increases the output of the analysis.

Methods and Materials

Generation of Replicates for Southern Blotting

Duplicate restriction reactions each containing approximately 3.6 μg PT7T3D (from Research Genetics) in a total reaction volume of 50 μl was digested with EcoRI and Notl at 37 °C overnight. This restriction reaction generates a MUCl clone 153986 (0.7 kb) insert and the linearized plasmid backbone (3 kb). The completed reaction mixtures were heated for 15 minutes at 80 °C to inactivate the restriction enzymes. The entire volume of each reaction (i.e., 50 μl) was loaded into separate wells of a 0.8% TAE agarose gel and run at 70 V until desired separation of the reaction products was achieved. One set of bands representing the linearized plasmid and the MUCl insert were separately cut from one lane of the gel and purified using Sigma Genelute Agarose Spin column kits for later use as probes. The remaining portion of the gel was soaked in denaturing solution (e.g., 1.5 M sodium chloride, 0.5 M sodium hydroxide) and in neutralizing solution (e.g., 0.1 M Tris, pH 8.0) for 15 minutes each. Thereafter, a capillary transfer was performed overnight using the following setup (from bottom to top): 10 x SSC transfer buffer, transfer paper, agarose gel, 10 T-T (#1-10) membranes (20/20 GeneSystems, Rockville MD), a nitrocellulose trap, 20-40 layers of transfer paper, and a standardized weight (30 g). Specific Hybridization to Membranes

After the transfer, individual membranes were pre-hybridized in 50% formamide, 5x SSC, 5x Denhardt's, 0.5% SDS, lOmM EDTA, 10 μg/μl salmon sperm DNA, 0.1 mM sodium phosphate, pH 7.0, at 50 °C for 60 minutes. Purified MUCl insert (approximately 27 ng) and linearized plasmid (approximately 30 ng) were labeled with ³³P using a random labeling kit from Amersham. The membranes were hybridized using 23-30 x 10⁶ cpm of labeled probe in 50% formamide, 5x SSC, 5x Denhardt's, 0.5% SDS, 10% dextran sulphate, lOmM EDTA, 10 μg/μl salmon sperm DNA, 0.1 mM sodium phosphate, pH 7.0, at 50 °C. Following overnight hybridization, the membranes were washed at room temperature twice for 15 minutes in 2x SSC, 0.5% SDS, and twice for 15 minutes in lx SSC, 0.1% SDS.

Results and Discussion Figure 13 shows that the MUCl insert was specifically detected on membrane layers 2 and 5, and the linearized plasmid was specifically detected on membrane layers 4 and 7 (other layers were examined and signal was observed). This example shows that multiple Southern analyses based on LES is a reliable, consistent method for studying several samples in one experiment. Different probes can be tested, each one against a different layer. The results can be normalized (not shown) by comparing the signals to one layer where an internal control such as actin or GAPDH is used. This approach allows for a high throughput Southern-blotting analysis of several genes in a single experiment, on the same samples. This is relevant not only for saving time and effort, but it is applicable in cases where the validation is performed on precious samples, such as human tissue specimens, where the yield of DNA is limited.

Example 4: In Situ Amplification and LES

This example describes methods wherein nucleic acids are amplified in situ prior to, during, or after transfer to replicate membranes. These methods are useful where, for example, nucleic acids of interest are present in quantities that could not otherwise be reproducibly transferred to, or detected on, LES membranes. These methods can be used to amplify nucleic acids within a tissue section or a gel, or nucleic acids that have already been transferred onto a membrane or membrane stack.

Standard methods of in situ amplification can be used to amplify nucleic acids in a sample, for instance a thin preserved tissue section, prior to transfer to LES membranes. By way of specific example, the following procedure can be used to perform in situ reverse transcription (RT) amplification (in particular, PCR) on an ethanol-fixed prostate tissue sample:

Slides containing paraffin preserved, ethanol-fixed prostate tissue section(s) are dewaxed by incubation in xylene for five minutes, twice. The slides are then submerged for 10 seconds in each of the following ethanol baths in sequence: 100%) ethanol, 95% ethanol, 70% ethanol, 95% ethanol,

100% ethanol. The slides are then submerged in xylene for 20 seconds and allowed to air dry prior to incubation for 10 seconds on a heating block set at 105 °C.

To remove DNA, the tissue is treated with DNAse; for instance, a standard DNAse incubation is carried out overnight. By way of example, 40 μl of RNAse-free DNAse solution (2 μl RNAse inhibitor, 30 μl DEPC H₂0, 4 μl DNAse, and 4 μl DNAse buffer) is added to the tissue section on the slide. A cover slip is placed on top, and bubbles carefully squeezed off the tissue. The slide is incubated overnight at 30 °C in a moisture chamber. After incubation, the slide is rinsed with IX DNAse buffer (in DEPC H₂0), then rinsed twice in DEPC water for 10 seconds each. The slide and sample is then gently air dried.

In situ reverse transcription is then carried out on the prepared tissue section. Sixty (60) μl of RT reaction mixture is prepared as follows:

12.0 μl 5X reaction buffer 8.0 μl 10 mM dNTP's 2.0 μl RNAse inhibitor 4.0 μl oligo dT primer 2.0 μl 20 U/μl superscript II

4.8 μl 0.1 M DTT 27.2 μl DEPC H₂0

The RT mixture is placed onto the prepared tissue section, which is covered with a cover slip while taking care to carefully squeeze bubbles off of the tissue area. The slides are then incubated for one hour at 42 °C in a moist chamber, and the reaction stopped by incubation on a heating block at 95 °C for two minutes. The cover slips are removed and the slides washed twice in DEPC water for 10 seconds. The slides are then air dried gently.

Amplification of the resultant cDNAs is carried out using standard techniques. For instance, Taq-based PCR amplification can be carried out on the tissue section, using PCR conditions

(temperatures, times, and cycle numbers) selected based on the products desired to be amplified. The selection of such conditions is well known to those of ordinary skill in the art; for instance, guidance is provided in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990).

By way of another example, one step in situ RT-PCR on a fixed and permeabilized sample can be performed using GeneAmp EZ rTth RNA PCR protocol in combination with GeneAmp in situ PCR system 1000 (Perkin Elmer) using the manufacturer's recommended procedure. This method consist of placing 40-50 μl of EZ RNA PCR buffer mix (50 mM Bicine; 115 mM potassium acetate; 8% (w/v) glycerol, pH 8.2; 300 μM dA, dG, dCTP; 165 μM DTTP; 135 μM labeled dUTP; 5-10U of rTth DNA polymerase; 2.5 mM Mn(OAc)₂; 0.45-1 μM of target-specific primers (or universal primers, or a combination of specific primers) onto a fixed and permeabilized sample on a microscopic slide, and sealing it with the silicone gasket and clip following the manufacturer's protocol (GeneAmp in situ PCR system 1000, Perkin Elmer). The sample is then placed in

GeneAmp in situ PCR machine and heated for 120 seconds at 94° C, and then amplified for 30-40 cycles of 94° C for 45 seconds, and 60°C for 45 seconds. After the amplification, the sample is washed and transferred to a membrane stack using methods described herein.

The resultant in situ RT-PCR amplified nucleic acids are then transferred out of the tissue section into at stack of membranes using one of the methods provided herein. For instance, the nucleic acids can be transferred by direct contact transfer. Alternatively, they are transferred first to a microtiter plate or other similar device (see, for instance, Example 5), and then transferred into a membrane stack. Once the nucleic acids are transferred into the stack, the individual membranes can be separated from each other and nucleic acid analysis performed as described.

Similar methods could be used to amplify nucleic acids within a gel prior to or concurrent with transfer to a membrane or membrane stack, or to amplify already transferred nucleic acids in situ on the membrane or membrane stack..

This example provides methods by which minute quantities of nucleic acids of interest can be transferred to, and detected on, replicate membranes.

Example S: Spin Transfer Amplification LES

This example describes another embodiment wherein nucleic acids present in a sample (e.g., a tissue section or gel) can be amplified prior to transfer to a stack of membranes. In this example, the nucleic acids of interest are physically transferred (by methods such as centrifugation or microdissection) into a multi-well plate where amplification of the nucleic acids takes place. The amplified products can be transferred directly from the multi-well plate to the membranes, using a variety of techniques.

An approximately 400 bp fragment of the human Papilloma virus type 18, E6 gene was amplified from the plus (+) control provided by Maxim Biotech for the HPV primers, using primers HPV-1017 and HPV-1018 obtained from Maxim Biotech. The amplification reaction (50 μl total volume) was carried out for 10 minutes at 94 °C, followed by 40 cycles of the following series of conditions: 45 seconds at 94 °C, 45 seconds at 60 °C and 45 seconds at 72 °C. Upon completion of the 40 cycles of amplification, the reactions were brought to 72 °C for 10 minutes, and then held until processing at 4°C.

Thirty (30) μl of the amplification reaction mixture was separated on a TB gel containing no EDTA, which gel was then run at 100 V for about 30 minutes. The gel was cut underneath the desired band and flipped upside down onto a 384-well plate so that the top surface of the gel faced the open wells of the plate. The lid of the 384-well plate and a foam gasket were used to seal the system, and a rubber band was used to hold the elements in place. The gel overlapped wells D15-L8 of the plate and, in particular, the 400 bp HPV E6 gene fragment overlapped wells F12-G12. The plate and gel combination was centrifuged for two minutes at 2,000 RPM using a Sorvall RT6000B Refrigerated Centrifuge (DuPont). The gel was then removed from the microtiter plate, leaving fluid in wells that had been "under" the gel.

One (1) μl of the contents of each of wells F15-F9 (corresponding to samples 1-7) and wells I12-D12 (corresponding to samples 8-13) was amplified using primers HPV-1017 and HPV-1018 under the same conditions as described above. To confirm that the individual wells contained nucleic acids of the expected size, 10 μl of each amplification reaction was loaded on a 1% agarose TBE gel. After electrophoresis, a 400 bp amplification product was identified in gel samples corresponding to wells F12, F13, G12 and H12 of the 384-well plate. Those wells match exactly the position of the original band transferred from the gel into the 384-well plate.

Optionally, in situations where the amount of nucleic acid in the gel (or contained in another sample, for instance a thin tissue section) is particularly low, the entire contents of each well of the microtiter plate is used for the amplification reaction, rather than removing a portion of the contents out into another reaction vessel.

In order to analyze samples that have been captured in the microtiter plate on a membrane stack, the amplified nucleic acids can be transferred from the microtiter plate into a gel-based medium, and then into the membranes of a stack. In one alternative method, a few microliters of warm agarose (e.g., 2% gel mix) or other non-polymerized matrix is placed into each well after the amplification reaction is completed, thereby forming a gel plug in each well. The resultant plate with gel plugs is then placed in contact with a stack of membranes and nucleic acids transferred out of the gel plate into the membranes. For instance, the plate with plugs is placed upside down, with a stack of membranes underneath it, and the assembly is spun in a centrifuge to transfer the nucleic acids into the membrane stack.

Optionally, a gel can be poured over top of the gel plugs (or a prepoured gel placed over the top of the plugs and optionally heated or otherwise induced to polymerize to the plugs), to generate a capping gel (usually very thin). This capping gel is then placed in contact with the surface of a membrane stack (optionally after being removed from the plate), and the nucleic acids are transferred into the gel using any of the methods described herein. The capping gel provides the benefit of enabling optional removal of the plate, so that the nucleic acids can be transferred into a stack of membranes using any of the procedures described herein, including both contact transfer and wicking transfer.

In yet another method, a thin gel (for instance, of 1-2% agarose) is poured in a size sufficient to be laid over the face of the plate holding the amplified samples. The nucleic acids are then transferred into the gel (for instance, using spin transfer). The resultant gel can then be placed in contact with the surface of a membrane stack and nucleic acids transferred into the stack as described herein.

The resultant membranes are then separated from each other, and analysis of the nucleic acid molecules on each membrane can be carried out as disclosed herein.

This example shows another method whereby nucleic acids of interest can be transferred to replicate membranes even where the nucleic acids in the sample are present in minute quantities.

Example 6: Characterization of Multiplex Northern Blots To assess technical capabilities of the LES nucleic acid transfer system, several performance parameters were examined, including hybridization characteristics, signal sensitivity, and reproducibility relative to standard blots. This example provides a description of these tests and their results. Materials and Methods

Hybridization and/or total RNA binding characteristics of the layered array membranes were assessed in several experiments as follows. Total RNA (15-30 μg) from cell lines MDA-MB-453 (Geneka Biotechnology Inc, Montreal Quebec), Jurkat (Geneka Biotechnology Inc, Montreal Quebec), HeLa (Ambion Inc, Austin TX), Osteosarcoma MG-63 (Ambion Inc, Austin TX) was electrophoresed in a 1%, denaturing agarose gel. After washing the gel twice for five minutes in DEPC-treated water, a standard northern blot capillary transfer was performed overnight, except that the ten-layer membrane system (20/20 GeneSystems) was substituted for a nitrocellulose membrane. The following setup was utilized (from bottom to top): 20X SSC transfer buffer, transfer paper (Gel Blot Paper, Schleicher & Schuell, Keene, NH), agarose gel, 10-layer membrane set (20/20 GeneSystems, Rockville, MD), one nitrocellulose membrane (Protran, Schleicher & Schuell, Keene, NH), 20-40 pieces of transfer paper (Gel Blot Paper, Schleicher & Schuell, Keene, NH), and a standardized weight (7 g/ cm²). After transfer, the membranes were UV-crosslinked (1,200 mJ), and total RNA capture was assessed by SYBR DX DNA Blot Stain (Molecular Probes, Eugene, OR). For tests to analyze specific gene levels, the membranes were pre-hybridized in 6X SSC, 0.5% SDS, 10 μg/ml salmon sperm, and 5X Denhardt's, at 55 °C for 30 minutes. Twenty-five to 50 ng of each probe was random- prime labeled using the Rediprime II Kit (Amersham Pharmacia Biotech, Buckinghamshire, England) and ³³P incoφoration. The membranes were hybridized using 2,000-10,000 cpm/μl, at 55 °C in a rotating tube overnight, and then washed two times for 10 minutes in IX SSC 0.5% SDS, and two times for 10 minutes in IX SSC, 0.1% SDS, and exposed using the Phosphorimager 445 SI (Microdinamic Engineering, Rockville MD).

Quantitation of signals was performed using ImageQuant software (IQMac vl.2). A set of parallel experiments was also performed under identical conditions, but the stack often layered membranes was replaced with a conventional nitrocellulose membrane (Protran, Schleicher & Schuell, Keene, NH). Stripping of layered membranes was performed in a boiling solution of 0.5% SDS for 5-10 minutes, and membranes were subsequently re-hybridized.

Results and Analysis

The quantity and size distribution of RNA that is captured by each of the membranes in the system was examined by transferring 15 μg of total RNA from MDA-MB-453 cells through the layers. As seen in FIG 17A, a typical rRNA pattern with both 28S and 18S bands is observed, indicating that the low binding capacity of the membranes results in rapid saturation during the transfer process, thus permitting the majority of the sample to progress through and bind to subsequent membranes. Measurement of total RNA content for each membrane is shown in Table 3, panel A. The maximal variability was in membrane #1 which showed a 32% increase over the average signal, and membrane #4 which showed a 23% decrease. This degree of alteration has minimal effect on subsequent probe hybridization results, and is within the normal range of experiment-to-experiment variability typically observed with standard northern blots.

Hybridization characteristics of the system were next characterized by analyzing an individual gene on each often membranes. FIG 17B shows a signal generated by a GAPDH probe on membranes 2, 4, 7, and 8 using 15 μg of total RNA from the osteosarcoma cell line MG-63. A specific 1.3 kb band corresponding to the GAPDH transcript is seen in each membrane. Quantitation of the signal is shown in Table 3, panel A. Similar to overall RNA levels, only a relatively small variation between the membranes was observed, ranging from a 41% decrease from the average to a 29%o increase. Taken together, the total RNA and GAPDH quantitation data show that the layered array system generates ten membranes that reliably bind RNA, and can be successfully probed for specific genes.

Table 3. Quantitation of signal variability on ten layered array membranes. Panel A: Inter- membrane signal variability on ten layered array membranes. Total RNA and GAPDH expression were compared to the average signal intensity (arbitrarily assigned as 1.0). Panel B: Infra-membrane signal variability on layered array blots. Each lane was compared to the average signal intensity for the probe listed (arbitrarily assigned as 1.0). For the HPV 18 and cdc2 probes, both the larger (a) and smaller (b) transcripts were analyzed.

In addition to its low capacity binding characteristics, provided membranes are designed to provide increased hybridization efficiency. Therefore, even though a membrane binds significantly less nucleic acid, the signal intensity after probing approaches that of a traditional blot. This feature significantly expands the utility of the system, as investigators can perform multiple blots from a single sample preparation, each with a high degree of sensitivity. To compare hybridization results between the layered array system and a standard blot, 15 μg of total osteosarcoma MG-63 cell line RNA was analyzed by both methods in parallel. After the transfer of RNA out of the gels, all membranes were probed, washed, and imaged under identical conditions. FIG 18A shows comparison of GAPDH signal on the nitrocellulose blot with a membrane from the array system. Qualitatively, the results were similar between the two blots in terms of specificity and hybridization background. Densitometric analysis indicated the GAPDH band on the new membrane had a signal equal to 90% of the nitrocellulose membrane. In general, we have observed that the layered array membranes produce band intensities ranging from 60-95% of traditional blots for both mRNA and proteins. The subset of membranes that show 40% less intensity have a minimal effect on the utility of the system; however, it is necessary in some experiments to expose the blots to autoradiography film for an extended period of time to produce band patterns that are identical to those seen on traditional blots.

The utility of standard Northern blots can be increased by stripping and re-probing them. While this approach is useful, it has significant limitations. The stripping procedure is harsh, typically resulting in decreased hybridization levels and increased background each time the blot is probed. Occasionally anomalous results are observed after this procedure, likely due to damage to the blot, and/or, alterations in hybridization characteristics of a subset of target molecules. Nonetheless, the ability to re-probe blots can be useful in many experiments. Therefore, a stripping procedure was evaluated this for the layered membrane system. FIG 18B shows hybridization, stripping, and re-hybridization to stacked membranes using GAPDH and actin probes. Although this procedure can be applied successfully to the layered membranes, a decrease in membrane performance was observed after the stripping procedure, similar to that seen with traditional blots. The use of the membrane array system eliminates the need for re-probing blots in many applications. To assess the uniformity and reproducibility of the layered array system, 20 μg of HeLa cell line total RNA was analyzed in triplicate using GAPDH, HPV- 18 E6 E7, PCNA, and cdc2 probes

(FIG 19). The selected target genes are present in HeLa cells at varying levels of abundance, thus the membranes could be evaluated across a 20-fold range of expression. As shown in Table 3, panel B, the signals for each lane were quantified and compared. The infra-membrane lane variability ranged from a 29% increase from the average to a 19% decrease, with a median variance of ± 9.3%. These results were then compared with the membrane variability of traditional northern blots using nitrocellulose membranes (Table 4). Twenty (20) μg of HeLa cell line total RNA was run on three separate electrophoresis gels and subsequently blotted onto nitrocellulose. The first blot was probed with HPV18 E6/E7, the second with PCNA, and the third with cdc2. As shown in Table 4, panel A, the signal intensity varied among the lanes from a 42% percent increase from the average to a 24% decrease, with a median variance of 13.6%. We also analyzed the inter-membrane levels of total RNA staining on the three standard northern blots, and compared it with data from the layered array membranes (see Table 3, panel A). As shown in Table 4, panel B, the inter-membrane total RNA levels among the three standard northern blots varied among the lanes from 42% percent increase from the average, to a 58% decrease. Therefore, based on both total RNA content and probe hybridization data, it is shown that the layered membrane array system performs similar to standard northern blots in terms of infra- and inter-blot reproducibility.

Table 4. Quantitation of signal variability on standard nitrocellulose blots. Panel A: Infra- membrane signal variability on standard nitrocellulose blots. Each lane was compared to the average signal intensity for the probe listed (arbitrarily assigned the value 1.0). For the HPV18 and cdc2 probes, both the larger (a) and smaller (b) transcripts were analyzed. Panel B: Inter-membrane signal variability on standard nitrocellulose blots. Each lane was compared to the average signal intensity of all nine lanes, and then the signal variance among the three blots was compared.

Table 4. Panel A

Probe Signal Intensity Probe Signal Intensity

HPV18-a* HPV18-b*

Lane 1 1.15 Lane 1 0.91

Lane 2 0.91 Lane 2 1.06

Lane 3 0.94 Lane 3 1.06

PCNA cdc2-a*

Lane 1 1.24 Lane 1 0.85

Lane 2 0.76 Lane 2 0.76

Lane 3 1.03 Lane 3 1.42

cdc2-b*

Lane 1 1.09

Lane 2 0.91

Lane 3 1.03

Table 4, Panel B

Probe Total RNA Signal Probe Total RNA Signal

Blot #1 Blot #2

Lane 1 1.12 Lane 1 1.42

Lane 2 0.62 Lane 2 0.42

Lane 3 1.18 Lane 3 1.18

Blot #3

Lane 1 0.82

Lane 2 1.03

Lane 3 1.18 The ability of the layered array system to reliably detect relatively small differences (2-3 fold) in gene expression levels was then evaluated, and compared data with that from a traditional blot. Thirty (30) μg of total RNA from Jurkat and MDA-MB-453 cell lines were separated on an agarose gel and transferred to a stack often membranes. PCNA probe was hybridized to membranes 2, 3, 5, 7, and 9, and GAPDH probe was hybridized to membrane 4. Relative PCNA expression between the two cell types was calculated using GAPDH levels to normalize the amount of RNA loaded on the gel. Each of the five membranes probed for PCNA showed higher levels of expression in the Jurkat cells, ranging from a 1.28 to a 2.69 fold difference. To compare these results with standard northern blots, two 30 μg aliquots of Jurkat and

MDA-MB-453 RNA were electrophoresed and subsequently transferred to two separate nitrocellulose membranes. Both blots were probed for PCNA and the band intensities normalized using total RNA levels in the gel. Both blots showed higher levels of PCNA expression in Jurkat compared to MDA-MB-453 cells. Blot #1 showed a 1.56 fold difference and blot #2 showed a 3.76 fold difference. Overall, the data indicate that both layered membrane arrays and standard northern blots can detect expression level changes at the 2-3 fold level, and show similar ranges of blot-to-blot variation.

Using the layered array system, typically one membrane is probed for a housekeeping gene to normalize gel loading, and expression measurements of additional transcripts are performed on the remaining layers. As a practical matter, this allows data to be generated quickly and efficiently. For standard Northern blots, investigators often normalize gel loading using total RNA levels in the gel and this is the method that was employed in the experiment described above for comparison. Alternatively, one can simultaneously probe a blot for a gene of interest and a housekeeping gene (if they are of different sizes), or probe for the gene of interest, strip the blot, and re-probe for a housekeeping gene. The layered array system was also compared with standard Northern blots using each of these approaches. In each instance, the layered membrane system performed as well as standard Northern blots in accurately measuring transcript levels.

Discussion The layered array system has been extensively tested and shown to be robust and reliable.

The methods described herein permit investigators to produce several (usually about 10) usable blots from each RNA gel, thus significantly increasing the utility of each gel. mRNA transcripts occasionally have a "granular" appearance after probing on layers 8-10. The reason for this artifact is not yet clear. Although these membranes may be considered less pleasing in appearance than standard blots, the granular appearance does not compromise accurate quantitation of band intensity. For a subset of RNA preparations, it is occasionally observed that the biomolecule content is less regularly distributed among the ten membranes, with a shift towards the first few layers. In these instances, the hybridization signal on membranes 1-3 often reaches 120-130% of that of standard blots, while membranes 8-10 can show levels equal to 40-50% of the otherwise expected amount. Quantitation of transcript levels on each blot is not compromised by this effect, and all ten blots produce useful data. The decrease in signal intensity for layers 8-10 can generally be overcome by using slightly more RNA, for example, 25-30 μg total rather than the usual 10-20 μg. If the amount of sample is limited and ten separate expression measurements are required, it is suggested to probe layers 1-7 for transcripts of low, moderate, or unknown abundance level, and to probe layers 8-10 for transcripts of higher abundance.

This disclosure provides methods of producing substantially similar replicates of nucleic acid samples, such as Northern and Southern gels, sequencing gels, and so forth. The disclosure further provides kits containing one or more components for use in the transfer and duplication methods. It will be apparent that the precise details of the methods described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

CLAIMSWe claim:

1. A method of detecting one or more nucleic acids in a sample comprising: providing a stack of at least two membranes; contacting the sample to the stack produce a loaded stack; maintaining the loaded stack under conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack, and allow capture of at least a portion of the nucleic acids on the membranes; and detecting at least one nucleic acid on one or more of the multiple membranes, wherein the nucleic acids in the sample are in defined two dimensional relationships relative to each other, and wherein the portion of the nucleic acids captured on the membranes substantially maintains at least a subset of those defined two dimensional relationships.

2. The method of claim 1, further comprising amplifying the nucleic acids prior to contacting the sample to the stack.

3. The method of claim 1 , further comprising amplifying the nucleic acids after contacting the sample to the stack.

4. The method of claim 1, wherein the method is a method of making multiple substantial copies of the nucleic acid content of the sample, and wherein the multiple membranes are the multiple substantial copies.

5. The method of claim 1 , wherein capture of at least a portion of the nucleic acids on at least one of the membranes is by direct capture.

6. The method of claim 1, wherein capture of at least a portion of the nucleic acids on at least one of the membranes is by indirect capture.

7. The method according to claim 1 wherein the membranes each comprise a porous substrate having a thickness of less than 30 microns.

8. The method according to claim 7 wherein at least one of the membranes comprises a material for increasing affinity of the membrane to nucleic acids.

9. The method of claim 8, wherein the material for increasing affinity of the membrane is coated on the at least one membrane.

10. The method of claim 7, wherein the porous substrate comprises a material selected from the group consisting of polycarbonate, cellulose acetate, polyester, polyethylene terephthalate, polyethelyle, polypropylene, and mixtures thereof.

11. The method of claim 8, wherein the porous substrate comprises polycarbonate.

12. The method of claim 8, wherein the porous substrate comprises polyester.

13. The method of claim 8, wherein the material for increasing affinity is selected from the group consisting of nitrocellulose, poly-L-lysine, and mixtures thereof.

14. The method of claim 8, wherein the material for increasing affinity comprises a nucleic acid-specific captor.

15. The method of claim 8, wherein the porous substrate comprises a polycarbonate substrate and the material for increasing affinity comprises nitrocellulose.

16. The method according to claim 1 wherein the sample is a RNA/Northern gel, a DNA/Southern gel, a nucleic acid sequencing gel, a tissue section, or a nucleic acid array.

17. The method of claim 1, wherein detecting the nucleic acids comprises: separating at least one membrane from the stack; and detecting at least one nucleic acid on the one or more of the separated membranes.

18. The method of claim 1 , wherein the conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack comprise passing a transfer liquid through the membranes.

19. The method of claim 1, wherein the conditions that permit movement of at least a portion of the nucleic acids onto membranes of the stack comprise providing a wick that facilitates movement of the nucleic acids through the stack of membranes in a desired direction of movement.

20. The method of claim 1, wherein the stack of membranes comprises 3 or more membranes.

21. The method of claim 20, wherein the stack of membranes comprises 5 or more membranes.

22. The method of claim 20, wherein the stack of membranes comprises 10 or more membranes.

23. The method of claim 1 , further comprising amplifying the nucleic acids in the sample.

24. The method of claim 23, wherein amplifying the nucleic acids in the sample comprises in situ amplification of at least one nucleic acid molecule prior to applying the sample to the stack.

25. The method of claim 23, wherein amplifying the nucleic acids in the sample comprises in membran-o amplification of at least one nucleic acid molecule.

26. The method of claim 1, wherein at least two of the membranes have differential binding affinities for nucleic acids.

27. A method of making multiple substantial replicate blots of a nucleic acid content of a sample, comprising: providing a stack of at least two membranes, wherein the membranes permit nucleic acids applied to a surface of the stack to move through the membranes, while capturing at least a portion of the nucleic acids on each of the membranes; and applying the nucleic acid sample to the stack, under conditions that allow the membranes to capture at least a portion of the nucleic acids from the sample, thereby making multiple substantial replicate blots of the nucleic acid content of the sample.

28. The method of claim 27, further comprising amplifying the nucleic acids prior to applying the nucleic acid sample to the stack.

29. The method of claim 27, further comprising amplifying the nucleic acids after applying the nucleic acid sample to the stack.

30. The method of claim 27, wherein at least a portion of the nucleic acids are captured on at least one membrane by direct capture.

31. The method of claim 27, wherein at least a portion of the nucleic acids are captured on at least one membrane by indirect capture.

32. The method of claim 27, wherein at least two of the membranes have differential binding affinities for nucleic acids.

33. The method according to claim 32, wherein the nucleic acid sample is a RNA/Northern gel, a DNA/Southern gel, a nucleic acid sequencing gel, a tissue section, or a nucleic acid array.

34. The method of claim 27, further comprising detecting one or more nucleic acids of interest on at least one of the multiple substantial replicate blots.

35. The method of claim 34, wherein detecting one or more nucleic acids of interest comprises exposing a plurality of the multiple replicate blots to at least one detector molecule.

36. The method of claim 34, further comprising separating the multiple membranes prior to detecting the nucleic acids of interest.

37. The method of claim 27, further comprising amplifying the nucleic acids in the sample.

38. The method of claim 37, wherein amplifying the nucleic acids in the sample comprises in situ amplification of at least one nucleic acid molecule prior to applying the sample to the stack.

39. The method of claim 37, wherein amplifying the nucleic acids in the sample comprises in membran-o amplification of at least one nucleic acid molecule.

40. The method of claim 1 , wherein the membranes have a high affinity but a low capacity for nucleic acids.

41. The method of claim 1 , wherein at least a portion of the nucleic acids are transferred to each membrane of the stack.

42. The method of claim 40, wherein the nucleic acids in the sample are separated on a solid support, and wherein the method comprises contacting the resultant separated sample to the stack.

43. The method of claim 42, wherein the nucleic acids are separated on a gel.

44. The method of claim 42, wherein the separation comprises electrophoresis.

45. The method of claim 44, wherein the electrophoresis is electrophoresis of a poly-A RNA sample.

46. The method of claim 45, wherein less than about 1 μg of poly-A RNA is loaded into a well of the gel.

47. The method of claim 46, wherein less than about 0.5 μg of poly-A RNA is loaded into a well of the gel.

48. The method of claim 46, wherein less than about 0.1 μg of poly-A RNA is loaded into a well of the gel.

49. The method of claim 46, wherein less than about 0.01 μg of poly-A RNA is loaded into a well of the gel.

50. The method of claim 44, wherein a sample comprising more than about 5 micrograms of total RNA is loaded into a well of the gel.

51. The method of claim 44, wherein a sample comprising about 10 to about 20 micrograms of total RNA is loaded into a well of the gel.

52. The method of claim 27, wherein the stack comprises at least 5 membranes.

53. The method of claim 52, wherein the stack comprises at least 10 membranes.

54. The method of claim 27, wherein each membrane is less than about 30 microns thick.

55. The method of claim 54, wherein each membrane is about 8 to 10 microns thick.

56. The method of claim 27, wherein at least one side of the membranes is treated to increase specific binding of the nucleic acids.

57. The method of claim 1 or 27, wherein the membranes are in a frame, the frame being mounted to the periphery of the membranes, wherein the frame defines a channel for passing fluids or air away from the space intermediate between the membrane and an adjacent membrane.

58. The method of claim 42, wherein the nucleic acids are amplified prior to contacting the stack to the separated sample.

59. The method of claim 58, wherein the nucleic acids are amplified within the solid support.

60. A kit for replicating a pattern of nucleic acids from a sample, comprising: a plurality of membranes, each having a coating on its upper and/or lower surfaces to increase specific binding of a target nucleic acid; a quantity of transfer buffer; and a fluid impervious enclosure.

61. A kit for uniquely visualizing a desired predetermined nucleic acid if present in a biological sample, comprising: a plurality of membranes, each having a specific affinity for at least one nucleic acid, and at least one detector species, adapted to detect the desired predetermined nucleic acid if bound to the membranes.