BACKGROUND OF THE INVENTION Coated filter material has been shown to be useful as a matrix for the isolation and archiving of nucleic acid. Applicants have recently shown that FTA coated glass microfiber can be used as a tool for the isolation and subsequent elusion of genomic DNA. The components of the FTA coating facilitate this procedure.

The FTA coating comprises several components that initiate cell lysis and enable nucleic acid binding. The coating also contributes to stability of the bound material.

The FTA coating functionally associates with the filter matrix or media. The coated filter material has been shown to be useful as a tool for the rapid isolation of nucleic acid from whole cells. For example, the United States Patents 5,496,562,5,756,126, and 5,807,527 all relate to FTA coated materials for the isolation of nucleic acid from whole cells. United States Patent Application 09/398,625, assigned to the assignees of the present invention, discloses an FTA coated glass microfiber used as a tool for the storage, isolation, and subsequent elution of genomic DNA from whole cell samples.

There are several problems associated with the filter media described in United States Patent 5,939,259. The media employs the use of toxic choatrophic salt to initiate cell rupture and nucleic acid binding. The use of a safe material and a material appropriate for obtaining cellular samples from, for example, delicate neonatal skin, and the use of a filter matrix that inherently binds nucleic acid and avoids the use of choatrophic salts to promote nucleic acid binding is desirable. Such a system would be quite useful and a significant improvement over the prior art.

It would also be useful to develop a filter material which enhances yields of eluted plasmid purification products from bacterial cultures. It would be desirable to obtain greater critical micelle concentration which thereby would generate greater lysing capability and greater yields of target nucleic acid.

Summary of the Invention In accordance with the present invention, there is provided a substrate for purifying nucleic acid consisting of a matrix and an anionic detergent fixed thereto.

The present invention further provides a method of preparing a filter matrix consisting of the step of fixing an anionic detergent on a matrix.

Further, the present invention provides a method for isolating and archiving nucleic acid by the steps of applying a nucleic acid sample to a substrate consisting of an anionic detergent fixed to a matrix whereby the substrate physically captures the nucleic acid. The nucleic acid is then bound to the substrate.

Brief Description of the Drawings Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: FIGURE 1 shows a Polaroid image of Ameolgenin PCR products from DNA isolated from a variety of filter media and analyzed on a 1.5% agarose gel stained with ethidium bromide, lane 1: genomic DNA isolation carried out using GF/C glass microfiber coated with 10% SDS (medium of the invention); lane 2: genomic DNA isolated using GF/C glass microfiber coated with 10% SDS/5.3M GuHC1; lane 3: genomic DNA isolated using GF/C glass microfiber coated with FTA (FTA Elutel. Iane 4: genomic DNA isolated using 31-ET cellulose coated with FTA; lane 5: no DNA PCR control; FIGURE 2 shows a UVP Gel Documentation image of an agarose gel showing the presence of isolated plasmid DNA from bacterial colonies using a variety of coated filter materials; the three-band pattern of elassic plasmid isolation being noted (supercoiled, nicked, and linear), lanes 1 and 2: Plasmid isolated using FTA coated GF/C glass microfiber (FTA Elute); lanes 3 and 4: Plasmid isolated using 10% SDS coated DBS1000 glass microfiber; lanes 5 and 6: Plasmid isolated using FTA coated glass flake impregnated glass microfiber; FIGURE 3 shows a UVP Gel Documentation image of an agase gel showing the presence of isolated plasmid DNA from liquid culture using a variety of coated filter materials in the GenSpin format; lane 1: 500ng commercially available pGEM plasmid DNA (Promega); lanes 2-5: Plasmid isolated using FTA coated GF/C glass microfiber (FTA Elute); lanes 6-9: Plasmid isolated using 10% SDS coated DBS1000 glass microfiber; and FIGURE 4 shows a UVP Gel Documentation image of an agarose gel showing the presence of different isolated plasmid DNA populations from liquid culture using a variety of SDS coated and FTA coated filter materials in the 96 well multiwell format; lane 1: 1% SDS coated DBS1000 presented with pGEM plasmid culture; lane 2: 2.5% SDS coated DBS1000 presented with pGEM plasmid culture; lane 3: 5% SDS coated DBS1000 presented with pGEM plasmid culture; lane 4: 7.5% SDS coated DBS1000 presented with pGEM plasmid culture; lane 5: 10% SDS coated DBS1000 presented with pGEM plasmid culture; lane 6: FTA coated GF/C presented with pGEM plasmid culture; lane 7: 1 % SDS coated DBS1000 presented with pUC19 plasmid culture; lane 8: 2.5% SDS coated DBS1000 presented with pUC19 plasmid culture; lane 9: 5% SDS coated DBS1000 presented with pUC19 plasmid culture; lane 10: 7.5% SDS coated DBS1000 presented with pUC19 plasmid culture; lane 11: 10% SDS coated DBS1000 presented with pUC19 plasmid culture; lane 12: FTA coted GF/C presented with pUC19 plasmid culture; lane 13: 1% SDS coated DBS1000 presented with pGEM. SPORT plasmid culture; lane 14: 2.5% SDS coated DBS1000 presented with pGEM. SPORT plasmid culture; lane 15: 5% SDS coated DBS1000 presented with pGEM. SPORT plasmid culture; lane 16: 7.5% SDS coated DBS1000 presented with pGEM. SPORT plasmid culture; lane 17: 10% SDS coated DBS1000 presented with pGEM. SPORT plasmid culture; lane 18 : FTA coated GF/C presented with pGEM. SPORT plasmid culture; MW: 1 kb ladder molecular weight markers (Life Technologies).

Detailed Description of the Invention Generally, the present invention provides a substrate for purifying nucleic acid, the substrate consisting of a matrix and an anionic detergent affixed thereto.

The source of the nucleic acid can be a biological sample containing whole cells. The whole cell can be, but not restricted to, blood, bacterial culture, bacterial colonies, saliva, urine, drinking water, plasma, stool samples, and sputum. The samples can be collecte by various means known in the art, transported to the substrate, and then applied thereto.

Alternatively, the substrate can be in the form of a sampling device, such as a swab, sheet material, or the like and the sample can be obtained directly from the source. In other words, the substrate can be in the form of a device which can swipe or otherwise obtain the cell sample from a source. The source can be a sample tube containing a liquid sample, an organ, such as a mouth, ear, or other part of a human or animal, a sample pool, such as a blood sample at a crime scene or the like, or other various sources of cells known in the scientific, forensic, and other arts.

The substrate consists of a matrix and an anionic detergent affixed thereto. The anionic detergent can be selected from the group including sodium dodecyl sulfate (SDS). SDS can be obtained in various forms, such as the C, 2 form and the lauryl sulfate. Other anionic detergents can be used, such as alkyl aryl sulphonates, long chain (fatty alcohol sulphates) olefine sulphates, sulphosuccinates, phosphate ester, sodium tetradecylsulphate and sodium 2-ethylhexysulphate.

Generally, 5%-10% SDS can be used in accordance with the present invention. For example, increased concentrations of SDS, up to 10%, which cannot be accommodated within an FTA cocktail, as set forth in the prior art patents discussed above, provided greater critical micelle concentration which generates greater lysing capability and thus greater yield of target nucleic acid, as demonstrated in the example section set forth below.

As specifically shown in Example 4, below, a definite optimum SDS concentration is achieved in the 5-7.5% SDS concentration range for coating particular glass microfiber in order to enrich for and purify different plasmid populations directly from liquid cultures in a multi-well format, such formats being well known in the art.

The matrix can be associated with the anionic detergent in different forms, well known in the coating art. For example, the anionic detergent can impregnate the matrix, such methods of manufacturing to cause impregnation being well known in the art. Alternatively, the anionic detergent can merely coat the matrix, such coating being obtained by methods well known in the art.

The matrix can be made from fibers well known in the filtration art. The matrix can include fibers selected from the group including cellulose, glass fibers, glass microfibers, poly (vinylidene fluoride) (PDVF), non-woven polyesters, and melt blown polypropylenes. The anionic detergent, such as the SDS, can be applied to the filter matrix at varying concentrations.

The fibrous filter matrix of the present invention can be manufactured in various forms. For example, the fibrous filter matrix can be manufactured in a sheet form, which allows for it to be in various formats such as multi-well plates, spin tubes, slides, cartridges, swabs, and pads.

The term substrate or matrix, as used above, most generally means a porous material or filter media formed, but not limited to, either fully or partly from the materials set forth above, and/or their derivatives. The matrix material does not inhibit the sorption of chemical coating solution and does not inhibit the storage, elusion, and subsequent analysis of nucleic acid containing material added to it. It is preferred that the matrix be of a porous nature to facilitate immobilization of nucleic acid. Similar to the disclosure in United States Patent Application 09/398,625 discussed above, the substrate of the present invention allows for elusion of the genetic material therefrom in a state that allows for subsequent analysis. Such elusion is a time efficient step thereby providing for almost immediately analysis.

By being fixed to the matrix, the anionic detergent is disposed, sorbed, or otherwise associated with the matrix of the present invention such that the matrix and anionic detergent coating function together to immobilize nucleic acid thereon through an action of cellular lysis of cells presented to the support. That is, the anionic detergent can be adsorbed, absorbed, coated over, or otherwise disposed, in functional relationship with the matrix.

The substrate of the present invention is capable of releasing the genetic material immobilized thereon to by a heat elusion step.

Preferably, such a heat elusion is accomplished by the exposure of the support having the genetic material stored thereon to heated water, the water being nuclease-free. This capacity to allow for elusion characterizes the various support materials of the present invention.

The matrix of the present invention is such that nucleic acid immobilized to it can remain so in a stable form and not exhibit degradation, shearing, endonuclease digestion, or UV damage. Accordingly, the substrate in the present invention can be used to transport and archive nucleic acid samples.

For the present invention to be useful in various arts, it is capable of immobilizing nucleic acid that is collecte in the form of a soluble fraction followed a simplifie elusion process during which immobilized nucleic acid is released from the matrix of the invention. The matrix of the invention yields nucleic acid of sufficient quality that it does not impair downstream analysis, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcripted mediated amplification (TMA), reverse transcriptase initiated PCR, DNA or RNA hybridization techniques, sequencing, and the like.

Nucleic acid immobilization to a solid support, although a suitable template for singular PCR reactions, cannot be measured or detected by traditional techniques such as optical density or fluorescence. Nucleic acid must be in solution for these techniques. Other post-purification techniques wherein nucleic acid is desired in the soluble form includes cloning, hybridization protection assay, bacterial transformation, mammalian transfection, transcription-mediated amplification, and other such methods.

The present invention provides nucleic acid in such a soluble form.

The present inventive substrate can be prepared by fixing an anionic detergent on a matrix. The fixing step can be achieved by either impregnating the anionic detergent within the matrix or coating the matrix, as discussed above.

The present invention provides a method for isolating and archiving nucleic acid by the general steps of applying a nucleic acid sample to a substrate consisting of the anionic detergent fixed to the matrix, the substrate physically capturing the nucleic acid, and then bonding the nucleic acid to the substrate. The bonding step is achieved by heating the substrate having nucleic acid applied thereto, by the method discussed above.

The applying step can be achieved by applying whole cells to the substrate. The substrate itself actually induces the lysing of the cells thereby releasing the nucleic acid into the substrate. By being a porous substrate, the substrate presents a vast surface area upon which the nucleic acid is bound.

A washing step, such as with various buffers set forth in the example section, but not limited thereto, can be achieved and is done after cell lysis. The substrate then physically captures the nucleic acid within the intrastaces thereof.

The bound nucleic acid can be released from the substrate for further processing and analysis. The release is achieved by washing steps at elevated temperature, as demonstrated in the examples below. Unexpectedly, as demonstrated in Example 2, enrichment for different populations of nucleic acid from the same cell source can be achieved using incremental temperature regimes. For example, as demonstrated in Example 2, plasmid DNA can be isolated and enriched from bacterial colonies using the substrate of the present invention. Populations, such as larger populations of supercoiled plasmid, followed by nicked plasmid and finally by linear plasmid migrating to the top of the isolating gel can be achieved utilizing incremental increases in incubation temperature.

It is known that the FTA coating cocktail of the prior art contains 2% SDS. It is unlikely that this percentage can be increased due to saturation points when in conjunction with other components of the cocktail. This limits the lysing capability of the FTA coating filters of the prior art as a critical micelle concentration of SDS can be easily reached when presented with large numbers of cells, such as with a bacterial colony. Therefore, substrates containing a greater concentration of the lytic agent, the anionic detergent, enable greater lysing capability and in turn, greater nucleic acid recoveries.

This is demonstrated in the examples set forth below.

The following examples demonstrate the preparation, function, and utility of the present invention in order to isolate nucleic acid from various sources. The examples further demonstrate the effect of SDS coating concentrations on glass microfiber for the purification of direct plasmids from direct liquid culture using a multiwell filter plate. Hence, the following examples demonstrate that the present invention can be used for the rapid purification and isolation of nucleic acids and genetic material from samples containing whole cells.

Examples Example 1. The Use of 10% SDS Coated Glass Microfiber For The Purification Of Genomic DNA From Whole Blood Samples: GF/C glass microfiber was cut to size and pieces soaked in a dish for one hour at room temperature in a solution of either 10% SDS (filter matrix of the invention) or 10% SDS/5.3M GuHC1. After soaking the wetted filters were placed in a convection oven and heated for thirty minutes at 80°C.

This soaking and baking process constitutes the coating process. To the coated filter materials, as well as FTA coated GF/C and FTA coated 31-ET cellulose, several drops of freshly finger stick drawn blood was spotted and allowed to air dry for two minutes. Once dried a 1 mm punch was taken from the dried blood spots and applied to individual 200ul polypropylene PCR tubes. To each tube containing a single 1 mm punch, 200ul of FTA Purification Reagent (0.29g NaCI, 5ml 1 M Tris pH 7.5,1 ml 0.5M EDTA, 2.5ml Triton X-1009 per 500ml) (Fitzco, Inc.) was added. Tubes were incubated for five minutes at room temperature with no shaking. Following incubation of the FTA Purification Reagent was aspirated from the tube. A second aliquot of 200ul of FTA Purification Reagent was added to each tube. The tubes were incubated for five minutes at room temperature without shaking.

Following incubation, the FTA Purifcation Reagent was aspirated from all the tubes. 200ul of TE (10mM Tris-HCI, 0.1 mM EDTA, pH 8.0) buffer was then added to each tube. The tubes were incubated for five minutes at room temperature without shaking. The TE buffer was fully removed from the tubes, leaving the now washed 1mm discs at the bottom of the respective tubes. 20ul of nuclease free water was then applied to every tube. Each tube was then administered to a thermal cycler and heated to 95°C for ten minutes. Following heat incubation the 20u1 of water was removed and dispensed into individual 25u1 Amelogenin PCR reactions. PCR amplification was carried out following parameters described by the manufacturer of the Amelogenin primer set (Promega). Following PCR 10ul of each PCR reaction was visualized on a 1.5% agarose gel stained with ethidium bromide, and photographed using a Polaroid camera.

It can be seen from the Amelogenin amplification results (Figure 1) that PCR product is noted in lanes 1 and 3. This illustrates that readily genomic DNA has been isolated and eluted from the filter media represented in these lanese Lane 1 is GF/C glass microfiber coated with 10% SDS (filter media of this invention) and lane 3 is FTA coated glass microfiber. We see that 10% SDS coated GF/C functions in terms of lysis, binding, and elution as well as the FTA coated GF/C. The advantage is that one does not have to manufacture the 10% SDS GF/C with the same level of control as the FTA coated GF/C which has multiple components within the FTA cocktail.

From Figure 1 it can be noted that PCR-ready genomic DNA has not been released from glass microfiber coated with a mixture of 10% SDS and 5.3M GuHC1 (lane 2), and FTA coated 31-ET cellulose (lane 4).

The non-appearance of PCR product in lanes 3 and 4 may be due to the inability of the filter material and coating to lyse cells. More probably, this effect is caused by an inability of the filter material and coating to release the bound genomic DNA after lysing (U. S. Patents 5,496,562,5,756,126, Lane 5 is a no DNA PCR control.

Example 2. Comparison of FTA Coated Filter Materials And A 10% SDS Coated Filter Material For The Purification Of Bacterial Plasmid DNA From Single Colonies: DH5a bacterial culture was transformed with the plasmid pGEM and streaked onto an LB medium petri dish. Following overnight incubation of the streaked petri dish at 37°C, individual bacterial colonies were picked and resuspended in 10u ! of PBS buffer. The resuspended colonies were then spotted to 7mm discs of either FTA coated GF/C glass microfiber, 10% SDS coated DBS1000 glass microfiber (the medium of the invention), and FTA coated glass flake impregnated glass microfiber. After spotting, the discs were placed into a 2ml Eppendorf tube. To each disc, 1ml of TE buffer containing 10ug/ml Rnase was added and incubated for five minutes at room temperature without shaking. After incubation, the TE/Rnase buffer was aspirated from the tubes and replaced with 1 mi of TE buffer an dincubated for five minutes at room temperature without shaking. Again, TE was aspirated and replaced with fresh TE buffer following incubation. Following the final TE buffer incubation, 200ul of nuclease free water was added to each disc and incubated at 65°C for fifteen minutes. Applicants have previously shown (lyer, M. et al. (2000) Rapid Plasmid Template Preparation using Novel Paper Based Systems. Abstract & Poster. Plant and Animal Genome Vlil Meeting, San Diego, California) that 65°C incubation preferentially elutes smaller plasmid DNA populations from FTA coated filter materials, enriching over larger bacterial genomic DNA populations that will be bound as a result of the initial lysis step upon cell contact with the coated filter material. Bacterial genomic DNA requires a higher temperature than 65°C in order to be released from the coated paper.

After heat incubation the 200ul fraction from each tube was removed and 20ul of it analyzed by agarose gel electrophoresis. The ethidium bromide stain agarose gel image was captured using UVP Gel Documentation System.

From the gel image in Figure 2 it can be clearly seen that plasmid DNA can be isolated and enriched for from bacterial colonies using coated filter media. The classic three-band appearance of plasmic on the gel is noted (larger populations of supercoiled plasmid, followed by nicked plasmid and finally linear plasmid migrating at the top of the tel). Of particular note is the greater yield apparent when 10% SDS coated glass microfiber is utilized compared to FTA coated matrices. FTA coating cocktail contains 2% SDS, and that this percentage is unlikely to be increased due to saturation points when in conjunction with other components of the cocktail. This probably limits the lysing capability of FTA coated filters as the critical micelle concentration of SDS can be easily reached when presented with large numbers of cells (such as a bacterial colony). Therefore, filters containing a greater concentration of the lytic agent (SDS) enable greater lysing capability and in turn greater nucleic acid recoveries. This example also demonstrates that the other components of the FTA cocktail are not required for efficient plasmid isolation. This means cheaper, simpler plasmid isolation filter and system could be manufactured.

Example 3. Direct Comparison of 10% SDS Coated Glass Microfiber And The FTA Elute Material Within The GenSpin Product For The Purification Of Plasmid DNA From Direct Liquid Culture 10ml of liquid LB media was inoculated with a single colony of DH5a bacteria transformed with pGEM plasmid. The liquid culture was incubated overnight at 37°C with constant shaking. After incubation 1ml aliquots of the grown bacterial culture were applied to the spin baskets of separate GenSpin tubes (Whatman, Inc.) and also GenSpin tubes which had had the original FTA Elute filter paper replaced with 10% SDS coated DBS1000 glass microfiber. Each tube type was carried out in quadruplicate.

Both sets of tubes were centrifuged at 12,000 xg for one minute, and the filtrate removed. To the tubes, 0.5ml of TE buffer containing 10ug/ml of Rnase was added and incubated for one minute at room temperature before centrifugation at 12,000 xg for one minute. The TE step was repeated twice more (without Rnase). After the final TE wash, 200ul of nuclease free water was added to the spin basket of each set of GenSpin tubes. Tubes were then incubated at 55°C for fifteen minutes in a water bath. Following heat incubation, the tubes were subjected to a final centrifugation step at 12,000 xg for two minutes. 20ul of the final filtrate was analyzed by agarose gel electrophoresis. The ethidium bromide stain agarose gel image was captured using UVP Gel Documentation System.

From the UVP Gel Documentation System image of figure 3, both sets of coated filter material in the GenSpin format purify and enrich for plasmid DNA populations within the liquid culture they were presented with.

Compared with Figure 2, heat incubation at the lower 55°C temperature produces more intact plasmid DNA (one supercoiled band noted). As seen in lanes 6-9, the 10% SDS coated DBS1000 glass microfiber has greater yields of plasmid DNA than FTA coated GF/C (FTA Elute material within the commercially available GenSpin), as seen in lanes 2-5. The 10% SDS coated material produce plasmid DNA that has a greater ratio of supercoiled DNA compared to the FTA coated material. This is illustrated by the presence of a single band.

By using an SDS coated filter material in the GenSpin format, Applicants have been able to purify high quality plasmid DNA directly from liquid culture without the need for cell harvesting, lysis, precipitation and binding that is required for traditional chaotrophic salt plasmid isolations.

Furthermore, the SDS coated material is more effective at purifying plasmid directly from liquid culture than FTA coated filter material.

Example 4. Effect Of SDS Coating Concentration On Glass Microfiber For the Purification Of Different Plasmids From Direct Liquid Culture Usina A Multiwell Filterplate Into the empty wells of an 800ul 96 well filterplate, discs of SDS coated DBS1000 glass microfiber were placed. The SDS concentrations of the placed discs were as follows: 1%, 2.5%, 5%, 7.5% and 10%. Also, some wells were filled with a disc of FTA coated GF/C glass microfiber (FTA Elute).

DH5a bacterial cells were transformed with either pGEM, pUC19, or pGEM. SPORT plasmids and grown overnight in separate 10ml LB cultures at 37°C with constant shaking. To each well of the filled multiwell plate, a 500ul aliquot of the bacterial culture was added. Addition of culture was carried out in such a way so that all the different filter materials under examination were presented with each of the bacterial cultures. After liquid sample application, the multiwell plate was placed on a vacuum manifold and vacuum applied for five minutes at-25inHg so that the wells could drain. With the vacuum remaining on, the following buffers were added in sequence: 0.5m ! TE containing 10ug/ml Rnase, 0.5ml TE buffer, and 0.5ml TE buffer. After the final TE buffer wash, 100ul of nuclease free water was added to each well under examination. The multiwell plate was then placed into a convection oven and incubated for fifteen minutes at 65°C. Following heat incubation the multiwell plate was placed on top of a multiwell collection plate and centrifuged at 1000 xg for two minutes so as to transfer the nuclease free water within the filter multiwell plate to the collection plate. 20u1 of the final filtrate was analyzed by agarose gel electrophoresis. The ethidium bromide stained agarose gel image was captured using UVP Gel Documentation System.

From the UVP Gel Documentation System image of Figure 4, it can be readily noted that all of the SDS coated and FTA filter materials enrich for and purify different plasmid populations directly from liquid culture in a multiwell format. There is a definite optimum SDS concentration for the coating on DBS1000 glass microfiber. It appears to be in the 5-7.5% SDS concentration range. At these concentrations, critical micelle concentration is maintained so that lysis is effective and yields high. At lower concentrations, it could be argued that critical micelle concentration is reached and lysis effectiveness is reduced. At higher SDS concentrations, the desired DNA may be damaged in some way such as denaturation. Applicants have therefore demonstrated an optimal SDS concentration for the coating of filter materials when used for the rapid isolation of plasmid DNA directly from liquid culture in a multiwell format.

Throughout this application, various publications, are referenced by author and year. Full citations for the publications are listed below. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.