Cards for storage and distribution of nucleic acid molecules

Abstract

A card having multiple tabs with absorbent or binding material onto which nucleic acid solutions can be spotted for storage and / or distribution to end users, such as customers who purchase nucleic acids. An aqueous solution of nucleic acid molecules is spotted onto the absorbent or binding material that is adhered to the tips of the tabs. Individual tabs can be cut or broken off from the card to recover the nucleic acid molecules. The card provides a convenient means of storing small aliquots of nucleic acid solution for weeks, months, or years. Methods for assembling the card, as well as methods for spotting, storing, and eluting nucleic acid molecules, are provided.

Description

[Technical Field]

[0001] The present invention relates generally to compositions for preserving nucleic acids in a format convenient for distribution to end users. The present invention further relates to methods for making, using, and storing cards suitable for adsorbing nucleic acids on such cards. [Background technology]

[0002] In recent years, the use of nucleic acid molecules has expanded, and distribution to researchers worldwide has become a routine requirement. However, storage methods for distribution have remained largely unchanged since the early days of nucleic acid isolation. Traditionally, specimens have been stored in aqueous solutions at subzero temperatures of -20°C or even -80°C. However, freezers take up space, have relatively limited capacity, require an uninterrupted power supply to operate, and sometimes fail with little warning. By extension, the need for electricity means that freezer storage is generally unsuitable for low-resource environments. Aqueous storage solutions are typically buffered to prevent other sources of damage to nucleic acids. Unbuffered water is harmful to nucleic acids due to pH fluctuations during freezing / thawing, and ice crystals formed during freeze / thaw cycles can shear molecules. Other chemicals often used in laboratory environments can be harmful to nucleic acids, especially over time, and should therefore not be included in any storage solution. For example, metal ions required for certain enzymatic reactions can damage nucleic acid molecules. Inadvertent contamination of aqueous solutions can also cause damage. Furthermore, nucleic acids in aqueous solutions are typically shipped in cryogenic packaging or even with dry ice, making distribution more costly and wasteful.

[0003] Alternatively, nucleic acids, particularly DNA, can be preserved by spotting an aqueous solution containing the nucleic acid molecule of interest onto a small piece of filter paper, such as Whatman® 3 mm chromatography paper (Merck KGaA; Germany), and allowing the spot to dry or the filter paper to dehydrate. Traditionally, molecular biologists use a pencil to draw a circle on a piece of filter paper and spot an aliquot of nucleic acid suspended in a buffered aqueous solution onto the circle. Each aliquot is allowed to dry until the desired amount of nucleic acid is dry within the circle before adding the next aliquot. In a dry or dehydrated state, nucleic acids, particularly DNA, are very stable, even at room temperature, especially when stored away from direct light. Dried, labeled filter paper strips stored in a box or other type of container allow for long-term storage for many years at ambient temperature under low humidity (less than approximately 60%) conditions. Dried blood samples that are more than 20 years old can be routinely used for assays such as SNP genotyping or PCR assays. See Sjoholm et al. (Clin Chem. 53(8) 1401 (2007)) and Cassol et al. (J Clin Micro. 30(12) 3039 (1992)). DNA plasmids are often transported as dried specimens on filter paper, which is generally considered a safe storage method; however, loss of DNA integrity has been reported over long periods (>3 months). See Murakami (Open Biotech 7(10) 2013). QIAcard® FTA® and FTA® Elute Card are commercially available cards for room-temperature collection, transport, storage, and purification of nucleic acids. Each card has up to four circles for applying nucleic acid samples and an area for applying a single label to the card. The dried sample area on the FTA® Elute Card must be punched to allow for up to four 3 mm punches to be transferred to a secondary container for nucleic acid recovery and / or amplification.

[0004] DNA molecules in solutions of the disaccharide trehalose have also been successfully dried in multiwell-format plates and found to be stable for at least two years at various storage temperatures. Alternatively, polyvinyl alcohol and various proprietary commercial reagents for dry DNA storage, such as Biomatrica® DNAstable® plates, are known, which protect DNA longer than trehalose and PVA at 56°C and room temperature, especially for diluted samples. However, DNA stored at -20°C yielded longer sequence reads and stronger signals, indicating that temperature is a crucial factor for DNA quality that must be considered, especially for long-term storage in solutions dried on substrates similar to multiwell plates. See Ivanova and Kuzmina (Mol Ecol Resour. 2013 Sep;13(5):890-8).

[0005] While spotting and labeling paper for nucleic acid storage is sufficient for managing a small number of nucleic acid species, there is still a need to produce large quantities of dehydrated samples for commercial production and distribution in a compact, traceable format. It would also be advantageous to have a format that is suitable for barcoding or QR coding, as is used for multi-well plates. It would also be advantageous to have a format that allows multiple nucleic acid species, such as multiple plasmid DNA vectors with different inserts or other elements, to be stored on a single piece of paper or card without concerns about cross-contamination between spots for each species. Currently, there are no products on the market that meet all of these needs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sjoholm et al. (Clin Chem.53(8) 1401 (2007)) [Non-patent document 2] Cassol et al. (J Clin Micro.30(12) 3039 (1992)) [Non-patent document 3] Murakami(Open Biotech 7(10) 2013) [Non-patent document 4] Ivanova and Kuzmina(Mol Ecol Resour.2013 Sep;13(5):890-8) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention is a card comprising a thin substrate with tab sections formed by perforations or tear lines. The flat top or bottom surface at the distal end of each tab is shaped to fit into a microtube with an absorbent material, such as paper, adhered to the surface of the card substrate. Nucleic acids can be applied to the absorbent material and stored and / or distributed to end users via mail or other means of delivery. [Means for solving the problem]

[0008] In one embodiment, the present invention provides a card for storing nucleic acid molecules, comprising a substrate of appropriate size and thickness having perforations forming a plurality of severable tabs, an absorbent material adhered to the flat upper or lower surface at the distal end of each severable tab, and a proximal region of each severable tab to which a label can be applied. The absorbent material may be pretreated to remove or inhibit agents that would damage nucleic acids. The absorbent material may be porous paper adhered to the substrate with a non-toxic adhesive. The card may be labeled by printing directly on the substrate or by applying an identifying label, such as a paper or plastic sticker. The label can individually identify the contents of each tab and identify the nucleic acid molecules absorbed into the absorbent material. The label identifying the card may also be printed or applied.

[0009] In another embodiment, the present invention provides a method of using a card for storing and / or distributing nucleic acid molecules to end users, comprising the steps of: providing a solid substrate having a plurality of severable tabs and an absorbent material adhered to the flat upper or lower surface of the distal end of each severable tab; applying an aqueous solution containing nucleic acid molecules to the absorbent material of at least one severable tab; allowing the aqueous solution to dry; printing or applying a label identifying the nucleic acid molecule applied to at least one severable tab; sealing the card in packaging; and storing the packaged card under appropriate conditions and / or distributing (e.g., by mail, courier, etc.) the packaged card to an end user. The appropriate conditions vary depending on the nature or type of nucleic acid spotted on the tab. Typically, the conditions are dry, protected from light or ultraviolet light, and may include refrigeration, freezing, or room temperature. Once spotted with nucleic acids, dried, and packaged, the card may be mailed or shipped to an end user.

[0010] Other features and advantages of the present invention will be set forth in the description of the invention which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The present invention will be realized and attained by the compositions and methods particularly pointed out in the specification and claims hereof. [Brief explanation of the drawings]

[0011] [Figure 1A] 1A-1D show diagrammatic views of the various components of a nucleic acid storage card. Figure 1A shows a thin plastic substrate 100 that has been cut, printed, and / or perforated (dashed lines 105) to form 16 pointed, detachable tabs 110. [Figure 1B] FIG. 1B shows absorbent material 115 adhered to each tab 110. [Figure 1C] FIG. 1C shows a label 120 that identifies each tab with a unique QR code to provide information about the nucleic acid spotted on the tab of the card. [Figure 1D]FIG. 1D shows the assembled card identified with a product label 120 that includes a QR code to identify the individual tabs. [Figure 2] FIG. 2 shows diagrams of nucleic acid storage cards with various configurations, including 20 tabs, 10 tabs, and 5 tabs. [Figure 3] 3 is a diagram of a cut-off tab 110 with absorbent material 115 attached to its distal tip and a microcentrifuge tube 20 for retrieving nucleic acids from the tab 110. Nucleic acids in an aqueous solution containing a neutral blue dye are spotted onto the absorbent material 115 and allowed to dry. The cut-off tab 110 is placed in a microcentrifuge tube 205 containing an appropriate amount of buffered elution solution. A change from clear to blue indicates that the dye and nucleic acids have been transferred from the tab into the elution buffer 210. [Figure 4] Figure 4 shows an exemplary card with a row of detachable tabs with absorbent paper adhered to the distal shaped end of each tab. Each tab is spotted with a solution of plasmid DNA spiked with a neutral blue dye. One tab is cut off and placed in a microcentrifuge tube. Each tab is labeled with an identifying QR Code® that provides a link to a description of the plasmid DNA. The card is also labeled with a QR Code® encoded with information describing the contents of the card. As shown on the card, the approximate size of the card is 3.5 inches wide by 2.5 inches high. [Figure 5] Figure 5 shows the color intensity of the eluate after 2 hours of elution from spotted tabs with nucleic acid solutions containing 2x, 5x, or 10x concentrations of dye: 4A shows xylene cyanol, 4B shows bromophenol blue, and 4C shows cresol red. [Figure 6]Figure 6 shows bacterial growth of E. coli transformed with plasmid DNA eluted from the tabs as shown in Figure 4. Xylene cyanol (XYLENE), which is toxic to E. coli, shows that no growth was observed. E. coli transformed with plasmids spotted with bromophenol blue (BROMO) or cresol red (CRESOL) for 2 hours showed similar amounts of growth. [Figure 7] FIG. 7 shows the growth of E. coli transformed with the successfully eluted plasmid in 120 μl of 1×TE buffer for 2 hours (6A) or 24 hours (6B). [Figure 8] Figure 8 shows the PCR products of DNA template and PCR primers stored on the card for 14 days at room temperature, eluted, and amplified. L=1 kb ladder. DETAILED DESCRIPTION OF THE INVENTION

[0012] The following description and examples detail some exemplary embodiments of the disclosed invention. Those skilled in the art will recognize that there are numerous variations and modifications of the invention that fall within its scope. Therefore, the description of a particular exemplary embodiment should not be considered as limiting the scope of the invention.

[0013] As used herein, the term "nucleic acid" refers to RNA, mRNA, genomic DNA, plasmid DNA, synthetic DNA or RNA, siRNA, and oligonucleotides.

[0014] As used herein, the phrase "nucleic acid protection properties" refers to properties that inhibit agents, compounds, enzymes, and / or environmental forces that damage nucleic acids. For example, moisture absorbed from a humid environment can damage nucleic acids over time. The presence of nucleases in the nucleic acid solutions or materials of the present invention can also cause damage. Nucleic acids should also be protected from metal ions, as these can promote the activity of contaminating enzymes. Thus, nucleic acid protection agents can include, but are not limited to, metal ion chelators, nuclease inhibitors, and / or dehydrating agents.

[0015] One embodiment of the present invention is a flat card comprising a solid substrate. The substrate may comprise a semi-rigid material capable of providing structural support for a test strip platform into which the substrate may be incorporated. The substrate may also be referred to herein as a card backer. The substrate may be a material such as plastic (e.g., polyethylene (PET), polyvinyl chloride (PVC), polyethylene terephthalate (PETG), polyimide, polycarbonate, polystyrene), ceramic, glass, paper, or a plastic-paper laminate, or any combination thereof. The material should be thick enough to be bent or cut to separate the individual tabs, typically in the range of 1 mil to 30 mils. A mil is a dimension equal to 1 / 1000 of an inch or 0.001 inch. Most human hair is 1 / 1000 of an inch or 0.001 inch. The most common size for thickness grades of plastic sheeting is 6 mils, which is 6 / 1000 of an inch or 0.006 inch. Generally, the thicker the plastic, the stronger the plastic. In some embodiments, the substrate may be metallic. The substrate may be pre-treated to be printable to obtain accurate color rendition on the substrate.

[0016] The card has multiple tab sections formed by perforations or tear lines. The distal end of each tab has absorbent material adhered to the flat top surface. The proximal region of each tab can be used to apply a label or barcode. The card can be any suitable size, but typically ranges from approximately the size of a credit card to the size of a 3-inch by 5-inch note card. The tabs can be arranged in parallel rows perpendicular to the long edges of the card, with absorbent material applied to the tabs along one long edge and an area for applying a label along the opposite long edge. The card can also have tabs located along both long edges of the card, along with a central spine area where an identifying label can be applied. The distal ends of the tabs can be shaped for insertion into microcentrifuge tubes or wells of a multiwell plate. For example, the distal ends can be tapered, pointed, or rounded. In one embodiment, the distal ends are square. The shape can be tailored to fit a particular type or style of tube. An advantage of the cards of the present invention is that their design prevents contamination from spots applied to adjacent areas. Prior art paper-based cards lack a physical barrier to prevent cross-contamination from capillary action or, if not properly cleaned, have the potential for nucleic acid carryover onto the hole punch. In contrast, the cards of the present invention provide a single, clearly defined absorbent pad that binds nucleic acid onto a substrate of non-absorbent material, creating a physical barrier between the tabs by eliminating continuous absorbent material. In addition, the tabs are designed to facilitate placement into and removal from elution tubes by having the plastic tabs near the top of the tube, distal from the absorbent pad, allowing them to be easily grasped with tweezers or other means commonly found in laboratories.

[0017] The absorbent material may be treated to remove or inhibit agents that would damage nucleic acids. The treatment may be a pretreatment, applied before adhering the absorbent material to a substrate, or it may be applied after the absorbent material has been attached to the substrate, i.e., as a spray or other liquid application to the absorbent material and substrate. Various agents, compounds, or inhibitors have nucleic acid protecting properties and are well suited for this application. For example, the absorbent material may be treated with a chelating agent to bind metal ions that would otherwise activate contaminating enzymes and damage nucleic acids applied to the absorbent material, especially over long periods of time. Chelating agents include, but are not limited to, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA), ethylenediaminetetraacetic acid (EDTA), murexide, dimercaptol, desferrioxamine, deferoxamine, enterobactin, and calbindin. Other agents that prevent damage are inhibitors of nucleases, such as DNase, which degrades DNA, or RNase, which degrades RNA. Meanwhile, one embodiment of a DNA storage card comprises an absorbent material that has been treated with RNase, free of DNase, to eliminate the possibility of RNA contamination. The use of water or other aqueous solutions treated with diethylpyrocarbonate (DEPC), diethyldicarbonate, diethyloxydiformate, or ethoxyformic anhydride is another RNA protection treatment. Yet another example is a dehydrating agent protection treatment to minimize contact of nucleic acids with water or moisture. Any of these treatments may be combined to protect nucleic acids and extend their useful shelf life.

[0018] The absorbent material is applied within the area delimited by the line at the distal end of each tab to form the nucleic acid application area. The application area of ​​each tab may be smaller than the distal end of the tab, particularly if the severable tab is straight rather than tapered, to physically separate each application area on the card from each other. The absorbent material may also be filter paper, such as acid-free cotton paper. Many types and brands of absorbent paper are known in the art, including Whatman® 3 mm chromatography paper (Merck KGaA; Germany). For example, Whatman® No. 1 paper, 180 μm or 0.18 mm thick, works well for this purpose. Other suitable examples include Hamilco white card stock, a 100 pound heavy cover card stock (Hamilco; Denton MD), Southworth 100% cotton paper stock 20 b / 75 GSM (Neenah, Inc; Atlanta GA), or Savoy 100% cotton paper 118 GSM (32 / 80 lb text). Other suitable materials include blotter paper, rayon filter paper, blanket qualitative filter paper, Whatman No. 1 paper, Schleicher & Schuell filter paper and electrophoretic paper products such as Kim-Wipe, SS-598, 2043a, and 593, nonwovens such as fiberglass and bonded polyester or bonded nylon, and other types of filter paper.

[0019] In one embodiment, the thickness of the absorbent material is determined by the thickness of the paper applied to the tab. In another embodiment, the thickness, length, and width of the absorbent material are all relatively equal, such that the absorbent material is cubic. An advantage of a cubic shape is that it can hold more nucleic acid molecules, such as a plasmid library of DNA molecules. For example, a card with a cubic shaped absorbent material can hold a library of plasmids, cDNA, or genomic DNA, which can serve as a template for PCR amplification of a single subunit of genetic material from within the library. The cubic shape may comprise a layer of absorbent paper or may comprise an encapsulating material formed from an absorbent powder. An exemplary cube is approximately 1 cm. 3of absorbent material, although other sized cubes, larger or smaller, are also contemplated.

[0020] Adhering other absorbent materials, such as absorbent powders, is also contemplated. For example, a glass silica matrix can form a DNA-binding vesicle when encapsulated in a "cage" that can be attached to a tab. The cage can be filled with untreated and / or treated materials that bind nucleic acids. Glass silica is an example of a material with inherent nucleic acid-binding properties; positively charged silica particles have a high affinity for the negatively charged nucleic acid backbone. Various support materials, including but not limited to silica, cellulose, agarose, or various plastics in the form of beads, slurries, or resins, can be treated with compounds to improve or impart nucleic acid binding activity. Examples include, but are not limited to, coupling diethylaminoethyl (DEAE) to silica beads or coupling arginine ligands to agarose.

[0021] There are many adhesives known in the art that are suitable for applying the absorbent material to the underlying substrate of the card without affecting the integrity of the nucleic acid applied to the absorbent material, and since the typical use of the eluted nucleic acid is the transformation of bacterial or eukaryotic cells, the adhesive should be non-toxic.

[0022] In one embodiment, the adhesive is a double-sided tape with pressure-sensitive adhesive exposed on both sides, allowing two parts to be bonded together with a carrier tape between the two parts. The carrier holding the adhesive can vary from a thin film of a fraction of a millimeter to a thick foam that helps dampen vibrations. Similarly, the adhesive can vary from a low-viscosity, repositionable adhesive to a permanent adhesive solution. Double-sided tapes with carriers can be manufactured with the same adhesive on both sides, or with different adhesives to meet the bonding requirements of different substrates. Suitable adhesives are typically those that bond well to plastics and / or metals. An example of a suitable double-sided tape is 3M™ Double Coated Tape 9495LE (3M Company, St. Paul, MN). Other examples of suitable adhesive double-sided tapes include, but are not limited to, proprietary adhesive tapes from 3M Company with part numbers 9310LE, 93015LE, 415, 93005LE, 9471LEm, 9457, 465, and 6035PC. The adhesive on the tape may include any type of polyester fiber, such as acrylic or polyvinyl acetate. Other adhesive types may be wet, contact, reactive, one-part, two-part, thermoset, hot melt, or pressure-sensitive adhesives. For example, the adhesive may be a liquid that is sprayed, painted, or otherwise spread onto the substrate, or it may be a solid, such as double-sided tape. Adhesives known as "super glues," including cyanoacrylate esters or similar polymers, may also be used. The use of epoxy, polyurethane, and polyimide adhesives is also contemplated.

[0023] Attachment of the absorbent material to the support may be accomplished in a variety of ways. A suitable method is by using a double-sided adhesive material. The adhesive material is laid as a layer on the support, and a tape liner is placed on top of it. Holes may then be punched, the tape liner removed, and the carrier secured with the adhesive surrounding the holes. Other suitable methods of attaching the carrier to the support include heat sealing and ultrasonic sealing. Yet another method is to place the carrier between two supports whose holes are generally aligned with each other. It will be understood that the attachment method is not limited to the methods described, as any non-toxic adhesive may be used according to the manufacturer's instructions, or any other method that achieves attachment of the absorbent material to the substrate.

[0024] Nucleic acids are typically stored in a clear buffer solution, such as 1x TE buffer. A liquid dye may be added to the aqueous solution of nucleic acids to visually indicate that the solution has been spotted onto the tabs. Neutral dyes are known in the art and are used to visualize DNA or RNA samples loading onto electrophoresis gels, including, but not limited to, bromophenol blue, xylene cyanol, and cresol red. Proprietary dyes, including SYBR™ Safe (Invitrogen; Waltham, MA) and EvaGreen® (Biotium; San Francisco, CA), are also known and may be used, but an inexpensive neutral loading dye is generally sufficient. By adding a neutral dye to the nucleic acid solution before spotting, a human or robotic optical system can visualize the colored dye to confirm that all tabs have been spotted. Additionally, migration of the dye into the eluate can be observed, providing an indication that the desired nucleic acid has been eluted from the tabs.

[0025] The present invention is particularly well suited for storing small aliquots of nucleic acids for extended periods of time, such as weeks, months, or even years. Accordingly, another embodiment is a method for using the card for nucleic acid storage by spotting aqueous solutions of nucleic acids onto individual tabs for storage and / or distribution. The aqueous solutions of nucleic acids may contain a dye that colors the absorbent material when the solution is applied and dried. After drying, the card may be sealed in a light-proof envelope or flat bag for storage. Storage may be at ambient temperature, under refrigeration, or in a freezer.

[0026] In another embodiment, the present invention is a method for distributing nucleic acid samples, such as sending nucleic acids to colleagues or customers who wish to purchase them. In addition to the labels on the individual tabs, the cards may have an identifying label, such as a number, a QR code, a barcode, or other type of label. Labels may be individually spotted on the tabs to identify the cards as containing a group of nucleic acid types, such as plasmid DNA vectors with various labeled inserts. Sealed envelopes or flat bags containing the cards may be distributed to end users by mail or any other conventional means of transportation. To recover nucleic acids from the cards, end users can tear or cut one or more tabs from the card. The tabs are inserted into microtubes containing a desired amount of elution solution, with the distal ends contacting the elution solution and allowing the nucleic acids to migrate from the absorbent material into the elution solution. The distal ends of each tab may be bent or pointed to allow the absorbent material to be completely immersed in the elution solution.

[0027] The card may have multiple tabs, each holding two or more types of plasmids, often 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more types of plasmids. Because the specificity of the PCR primers determines the type of amplified product, tabs holding multiple types of plasmids are particularly well suited as template pools for PCR amplification. While a cube may be capable of storing 1,000 or more plasmids or other nucleic acid libraries, any embodiment of the card can store multiple nucleic acid molecules. In one embodiment, the cube contains 500 unique plasmid DNA molecules. The accompanying card has tabs, each with a unique PCR primer pair adsorbed on it. These PCR primer pairs can be used to amplify a specific gene sequence found in one of the 500 plasmids on the cube.

[0028] Before exemplary embodiments of the present invention are described in more detail, it is to be understood that the present invention is not limited to any specific embodiment described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values ​​is provided, unless the context or description clearly indicates otherwise, it is understood that each intervening value (to the tenth of the unit of the lower limit) between the upper and lower limits of that range is included in the range and encompassed by the invention. In addition, unless the context or description clearly indicates otherwise, smaller ranges between any two values ​​within the range are also included.

[0030] Unless defined otherwise, 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. Representative exemplary methods and materials are described herein; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0031] All publications and patents cited herein are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual date of public availability, which may need to be independently confirmed.

[0032] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, it should be noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to support the use in the claims of exclusive terminology such as "solely," "only," etc. in connection with the recitation of claim elements, or "negative" limitations such as "absent from [the particular feature or element]," or "except for [the particular feature or element]," or "the particular feature or element is not present (e.g., not included)...."

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein, having distinct components and features, may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events described or in any other order that is logically possible. [Example]

[0034] The following examples provide exemplary compositions and methods for making and using the nucleic acid storage cards of the present invention. These examples describe materials and methods using the embodiments shown in Figures 1-7. Further details are provided in the "Brief Description of the Figures" section.

[0035] Example 1 Nucleic Acid Storage Card Components FIG. 1A shows a card 100 with severable tabs 110 defined by lines 105 that may be printed and cut with scissors, or perforated and torn or broken off to remove the desired tab. The distal ends of the tabs are cut to form pointed tips. In this exemplary embodiment, the card is comprised of 16 tabs. FIG. 1B shows the distal ends of the tabs, each with a pointed shape, with a triangular piece of absorbent material adhered to it. FIG. 1C shows a label 120 that identifies each tab with a unique QR code. The central region of the label 120 can be printed with a label that identifies the entire card. FIG. 1D shows an assembled card 101, identified by individual tabs 110 with a product label 120 and a QR code that identifies the card. Each tab's unique QR code links to a website containing information about the nucleic acid molecules spotted on that tab. The information may include, but is not limited to, RNA or DNA maps, sequence files, links to references, descriptions of various elements such as regulatory elements, promoter identification, insert identification, primer locations and sequences, restriction enzyme sites, and antibiotic resistance genes for selective growth on culture plates or in liquid culture.

[0036] FIG. 2 shows diagrams of cards having various numbers of severable tabs, including 20, 10, or 5.

[0037] Example 2 Use of neutral dyes to indicate the presence of nucleic acids Figure 3 shows a card with 10 detachable tabs spotted with a nucleic acid solution containing a neutral blue dye, with one tab cut off and placed in a microcentrifuge tube, ready for the addition of elution fluid.

[0038] Example 3 Transfer of nucleic acids from the tab to aqueous solution Each portion of the absorbent material can be spotted with a different sample of nucleic acid molecules, such as a plasmid vector carrying a recombinant DNA insert or clone. Figure 4 shows a cut-off tab pre-spotted with an aqueous solution of plasmid DNA containing a neutral blue dye. A QR code® is attached to the tab to allow the end user to identify the plasmid DNA. The cut-off tab is placed in an elution buffer in a microtube. The blue neutral dye provides a visual indicator of the transition from the absorbent material to the elution buffer. After elution, the tab is removed and discarded. The label on the tab is a plastic sticker that can be peeled off and affixed to the microtube before disposal to identify it as eluted plasmid DNA.

[0039] Figure 5 shows the co-migration of plasmid DNA spotted onto the card tabs at 2x, 5x, or 10x concentrations with neutral dyes, where 1x is equivalent to 1 µl of dye added to 50 µl of TE buffer. 5A shows xylene cyanol, 5B shows bromophenol blue, and 5C shows cresol red.

[0040] Example 4 Integrity of nucleic acids eluted from the tube Figure 6 shows the growth of bacteria transformed with the kanamycin-resistance plasmid DNA eluted in Figure 5 and plated on agar plates supplemented with ampicillin. Xylene cyanol was toxic to the bacteria, and no colonies were observed. Growth was abundant on plates using bacteria transformed with plasmid DNA recovered from tabs spotted with solutions containing bromophenol blue or cresol red, indicating that the plasmid was intact and the dyes were appropriately nontoxic.

[0041] Example 5 Testing the effects of different adhesives on nucleic acid integrity and bacterial toxicity Some adhesives may damage nucleic acids or contain components that are toxic to bacteria. Figure 7 shows the growth of bacteria transformed with kanamycin-resistant plasmid DNA spotted onto absorbent material adhered to a plastic substrate with various adhesives. The adhesives used were 7A: 3M™ 9495LE, 7B: 3M™ 93010LE, 7C: 3M™ 93015LE, and 7D: 3M™ 415. Aliquots of kanamycin-resistant plasmid DNA suspended in TE buffer were spotted onto the tabs, allowed to dry overnight, and eluted from the absorbent material. The eluate was used to transform E. coli and spread onto agar plates containing ampicillin. All four plates showed good recovery of plasmid DNA with ample growth, indicating nucleic acid integrity and the absence of toxic contaminants carried over from the adhesive.

[0042] Example 6 Rapid card production and use A 200-mil thick card backer material is used to form large quantities of cards using an automated system that prints, processes, and feeds through a paper gluing station. The cards are then cut to the desired size and shape for various applications. Each card produced can have multiple styles, from perforated tabs to self-snipping or break-off / tear-off tabs, creating a user-friendly method for removing each tab.

[0043] DNA plasmids are spotted onto the tabs, one plasmid per tab. A label may be printed on each card during the manufacturing process. Alternatively, the cards are "blank" manufactured without labels, with dedicated peel-and-stick labels applied to the cards later. The labels are used to identify the card type, i.e., each carrying a specific set of plasmids. A secondary label printed or applied to each tab in the form of a QR Code® identifies the specific plasmid spotted on each tab. Each QR Code® encodes an annotated map of the associated DNA plasmid and a link to instructions for elution and propagation, or a user-specified link to the data.

[0044] The cards may be cured and individually sealed in Mylar envelopes that also have labels attached to identify the card type and plasmid. The packaged cards are stored at room temperature or in other controlled environments until mailed or shipped to the end user. The user can identify the card and plasmid using the label and QR code along with instructions for use.

[0045] Example 7 PCR primer storage on cards The cards are useful for storing oligonucleotides, such as those used as PCR primers. The oligonucleotides may be stored as a single type of oligonucleotide (i.e., a single forward or reverse primer), in pairs (i.e., a pair of forward and reverse primers for a specific template), or in groups of pairs (i.e., pairs of forward and reverse primers for various templates). Furthermore, they may be stored on a single tab, or as an array of single tabs, an array of tab pairs, or an array of multiple tabs.

[0046] Example 8 Amplification of the on-card stored template library using on-card stored primers Five different pDNA species were spotted onto individual tabs on the card as single templates and stored. A group of all five DNA species was spotted onto a sixth tab and stored. Specific primer pairs were synthesized and spotted as pairs onto separate cards. The cards were stored at ambient temperature for 14 days, and the contents of each tab were eluted with 100 microliters of TE buffer. PCR amplification was performed with various combinations of eluted templates and primer pairs. As shown in Figure 8, samples of the combinations shown in Table 1 were electrophoresed in an agarose gel. Proper amplification was demonstrated in lanes 1 and 4 for DNA1 and DNA2, as well as in lane 10 for the combination of DNA1 and DNA2, and in lanes 11-13 for the combination of all five templates, DNA1-DNA5. This example demonstrates that the integrity of the reactions was maintained during safe storage. [Table 1] Table 1. Template DNA and primer pair combinations for PCR amplification

[0047] While the present invention has been described in terms of exemplary embodiments thereof, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments precisely as described above, but should also include all modifications and equivalents of those embodiments within the spirit and scope of the description provided herein.

Claims

1. A card for the storage of nucleic acid molecules, comprising: a flat solid substrate having perforations forming a plurality of severable tabs; an absorbent material adhered to the upper or lower surface of the distal end of each of said severable tabs; a proximal region on each of the severable tabs to which a label can be applied; consisting of A card characterized by:

2. the flat solid substrate is selected from the group consisting of plastic (e.g., polyethylene (PET), polyvinyl chloride (PVC), polyethylene terephthalate (PETG), polyimide, polycarbonate, polystyrene), ceramic, glass, paper, or plastic-paper laminate, or any combination thereof; 2. The card of claim 1.

3. the absorbent material is treated with at least one agent having nucleic acid protecting properties before being adhered to the upper or lower surface; 4. The card according to claim 3.

4. the at least one agent is selected from the group consisting of a dehydrating agent, a nuclease inhibitor, and an ion chelator; 4. The card according to claim 3.

5. The absorbent material is porous paper.

2. The card of claim 1.

6. The absorbent material is adhered with an adhesive.

2. The card of claim 1.

7. The adhesive is Pressure-sensitive double-sided tape, Including, 7. The card according to claim 6.

8. The label is applied by printing or by applying an identification sticker.

2. The card of claim 1.

9. the label identifies the nucleic acid molecule absorbed by the absorbent material; 7. The card according to claim 6.

10. one or more nucleic acid molecules are absorbed to the absorbent material of one or more of the cleavable tags; 2. The card of claim 1.

11. 1. A method for storing nucleic acid molecules and / or distributing nucleic acid molecules to end users, comprising: providing a card comprising a flat, solid substrate having at least one severable tab and having an absorbent material adhered to an upper or lower surface of a distal end of said at least one severable tab; applying an aqueous solution containing one or more nucleic acid molecules to the absorbent material on the at least one severable tab; drying the aqueous solution; printing or applying a label to said at least one detachable tab that identifies said nucleic acid molecule; sealing the card in a packaging material; storing the packaged cards under appropriate conditions and / or distributing the packaged cards to end users; Including, A method characterized by:

12. The suitable conditions are selected from the group consisting of room temperature, refrigeration, and freezing. The method of claim 11.

13. mailing or shipping the packaged card to the end user; Including, The method of claim 11.

14. 1. A card for storage of a library of nucleic acid molecules, comprising: a flat solid substrate; a distal end having a cube of absorbent material adhered to the substrate; a proximal end having a label applied to the substrate; Equipped with A card characterized by:

15. a library of nucleic acid molecules is absorbed into the cubic absorbent material; 15. The card of claim 14.

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