The present invention relates to printed materials comprising a support having an oligomer and/or polymer applied thereon, a method for preparing the same and a method for delivering and storing the oligomer and/or polymer.
A scientist consulting a scientific publication or article relating to an interesting clone of a molecular substance (e.g., genomic DNA, cDNA, RNA, mRNA and PNA) would sometimes like to use this clone for making experiments. In order to obtain the clone, he needs to contact the author of the publication and start a series of formality procedures necessary to obtain it. In some cases, the address contained in the publication has changed, or the scientist has moved to another laboratory and as a consequence it takes a long time to obtain the clone. In some cases, the vector or cell comprising the interesting sequence is no longer available and the scientist has no prompt availability of it and needs to make it again.
After the scientist makes a request for the molecular substance of interest, the molecular substance is sent to him according to the delivery systems known in the art, for example, in the form of a tube. The molecular substance of interest can also be delivered upon request in form of sheet-like supports on which RNA, PNA, other high molecular fragment or DNA is fixed or printed (U.S. Pat. No. 6,258,542). However, all these delivery systems known in the art require a request from the interested receiver, the preparation of the requested molecular substance in a form suitable for dispatching, manufacturing of the device suitable for delivery and delivery it. Further, the delivery may take several months.
The present invention solves the problem in the art by providing a printed material from which at least an oligomer and/or polymer can be obtained immediately and directly, without need to make a request for it.
The invention also provides a method for preparing such a printed material.
The invention further provides a method for delivering and/or storing an oligomer and/or a polymer.
1: Printed material
2: Support
3: Spot of oligomer and/or polymer (high molecular substance)
4: Spot of oligomer and/or polymer (low molecular substance)
5: Grid line
6: Perforated line
7: Hole
8: Pocket
9: Slit
10: Binding ring
11: Cover
12: Loose-leaf sheet
20: Printed letters containing an oligomer (a low molecular substance)
21: Printed letters containing an oligomer and/or polymer
30: Barcode
Various embodiments of the printed material of the present invention are hereinafter explained by reference to the drawings.
The article is in the form of a folded leaflet. The printing paper of the article is a support (2) on which the oligomer and/or polymer is applied. The printing paper has the spots of the oligomer and/or polymer in the centers of grids of a checker-board pattern. If a scientist reads this article and wants to make an experiment using the oligomer and/or polymer, he can obtain this substance immediately by cutting off a strip with the desired spots from the leaflet along grid lines (5) and eluting the oligomer and/or polymer from the strip. When the oligomer and/or polymer is a nucleic acid, it can be advantageously recovered by amplification techniques known in the art, for example PCR by using the proper primers. These primers may be included in the same support or in a separate support of the printed material. The nucleic acid recovered can also be transferred into a prokaryotic or eukaryotic host cell, for instance E. coli, according to known techniques, for example Sambrook et al., 1989. This article allows the reader to obtain the oligomer and/or polymer easily and immediately after he or she has read it. It is not necessary for the reader to contact the author or a depository institution having the oligomer and/or polymer and wait long to get it in his or her possession.
In a modification of the printed material shown in
The use of the bar code is not limited to this embodiment but can used in any support according to the present invention.
When an oligomer and/or polymer is fixed as spots on a support, a strip having one or more spots can be cut off with scissors, a cutter and the like, the strip can be transferred to a micro-test tube, and the oligomer and/or polymer may be eluted from the strip and amplified under ordinary conditions by polymerase chain reaction.
In this case, one or more primers for polymerase chain reaction may be supplied in the form of dots, or powder or liquid
The primer(s) in the form of spots may be placed anywhere on any page, or may be added at the end of the printed material as an appendix.
Even if there are various types of oligomer and/or polymer, it is also possible to supply a primer in the form of liquid or dry powder by containing such primer in an ampoule or a small test tube in such a case where a common primer may be used in a certain extent so that the number of types of primer may be reduced. The nucleic acid can also recovered by transferring it into a plasmid, a vector and/or an eukaryotic or prokaryotic host cell, for example E. coli or the like, according to known technique (for ex. Sambrook et al., 1989). The nucleic acid can be maintained and stored in this form until next use.
The present invention discloses a method for preparing a printed material comprising at least one support having at least one oligomer and/or polymer applied thereon, comprising the step of applying the oligomer and/or polymer on the support before or after printing the material. The oligomer and/or polymer can be fixed or printed on the support according to known techniques.
The present invention also provides a method for delivering and/or storing at least one oligomer and/polymer applied on at least one support comprised in the printed material, comprising the step of i) applying the oligomer and/or polymer on the support before or after printing the material, and ii) delivering and/or storing the printed material comprising the oligomer and/or polymer.
When the oligomer and/or polymer is nucleic acid, the present invention discloses a method for storing a nucleic acid by providing a printed material and/or a support comprising nucleic acid applied thereon, recovering the nucleic acid by transferring it into a host cell, and storing it.
The present invention therefore discloses a method for delivering biological molecules comprising the steps of applying at least one nucleic acid on the support before or after printing of the material, delivering the printed material, recovering the at least one nucleic acid by elution, amplification and/or transferring it from the support into a host molecule. In case the biological material applied on the support is a plasmid comprising cDNA, the plasmid is recovered from the support. Then the plasmid is subjected to amplification according to known technique, for instance PCR, and the cDNA is amplified. Then an electrophoresis gel is carried out (ex. Sambrook et al., 1989) and the cDNA can be recovered from the gel and used for any purpose. As said above, the amplified cDNA can also be "stored" into a host cell and kept in that form until next use. The DNA included in the host cell can also further delivered in this form.
The invention also provides a printed material comprising part of or all the substances necessary for an experiment, for example nucleic acid, primers, enzymes and/or solutions like buffers. All these molecules or substances can be applied on the support and then recovered by the reader or receiver, so that he can immediately carrying out the experiment and does not need to request the single substances, measure the concentrations of the substances and prepare them.
The invention therefore also relates to a method for providing, delivering and/or storing the oligomer and/or polymers necessary for carrying out an experiment on a printed material.
This embodiment is however not limited to printed material. Part or all the substances necessary for carrying out an experiment, for example nucleic acid, primers, enzyme and buffers can be applied on a single support, like a card or a sheet. Accordingly, the present invention also relates to a single support comprising more than one or all the substances for carrying out an experiment. Further, the invention provides a kit for carrying out an experiment comprising more than one or all the substances for carrying out an experiment applied on a support. The more than one substance can be nucleic acid, primer(s), enzyme(s), buffer, other solutions and the like. Preferably, all the substances necessary for carrying out the experiment can be added on the support. The support can be paper, card, sheet, and the like, as described in any embodiment of the present invention. Preferably, the kit comprises more than one substance and solution for carrying out an experiment applied on a water-dissolvable paper (for instance Mishima paper) according to the invention.
The support according to the invention can also be a wafer, as described above. The invention therefore related to a printed material comprising oligomer and/or polymer applied on a wafer and to a method for delivering and/or storing oligomer and/polymer by providing a printed material comprising a wafer having oligomer and/or polymer applied thereon.
Further, the present invention also provides a wafer comprising a oligomer and/or polymer and to a method for delivering and/or storing oligomer and/or polymer by applying oligomer and/or polymer on a wafer and delivering and/or storing it. The oligomer and/or polymer can be recovered by dissolving the wafer into water. Furthermore, the present invention also provides a method for synthesizing cDNA, exons and preferably full-length cDNA, from genomic DNA. The method is carried out from the genomic DNA comprising one specific gene, for example human luteinizing hormone (hLH) (however the method is not limited to the preparation of the full-length FL or exon(s) of this gene but any gene or exon can be prepared).
The starting material is the whole genomic DNA of a cell, for instance a human cell. Genomic DNA can be purchased (for instance from BD Bioscience Clontech, US) or prepared with standard technology. A source of genomic DNA can be any biological material obtained from a patient, for example blood. For the purpose of the present invention the genomic DNA in any form, including genomic DNA prepared from blood, fluid, liquid, or any other biological material or even purchased or prepared in purified form will be here also indicated as "template" or "template DNA".
An example of the realization of this method for the preparation of FL-hLH (full-length human luteinizing hormone), which is constituted by two exons, is shown in FIG. 19 and Example 8. A set of primers capable to amplify and/or ligating the exons of the desired gene are used. The set of primers is composed of a pair of primers capable of hybridizing with each exon. One primer of a first pair of primers hybridizes with the first exon and at the same time also partially hybridize with the extremity of the other (next) exon. For instance, in
As it is shown in FIG. 19 and Example 8 a plurality of set of primers can be used of the preparatio of cDNAs from template DNA. In the example, the pair of primers for amplification of exon 1, the pair of primers for amplification and exon 2 and the set of primers (therefore comprising all the set of primers) for the amplification and ligation of the exons can be spotted in the same support. In this case, not only the full-length of the gene but also one or more exons will be synthesized (as shown in FIG. 20). Accordingly, the present invention also provides a printed material and/or support comprising at least one set of primers applied thereon, this at least set of primers capable of synthesizing a FL-cDNA from the genomic DNA. This set of primers comprises primers for the amplification of the exons of a gene comprised in the template DNA and primers for the ligation of the amplified exons into a FL-cDNA. The printed material and/or support may also comprise one or more primers for amplifying one or more exons. Preferably, the printed material and/or support may comprise a set of primers for the synthesis of FL-cDNA and optionally further set(s) of primers for the amplification of one or more exons.
The printed material and/or support may further comprise one or more enzyme catalyzing these reactions (for instance Taq polymerase) and/or any solution necessary for carrying out the experiment, for instance a buffer solution.
The reader or receiver of the printed material and/or support can therefore recover the at least set of primer(s) from the printed material and/or support and add the template DNA, enzyme and buffer. In case the enzyme and buffer are also applied on the printed material and/or support, the receiver can recover all the elements from it, without need to obtain the enzyme and buffer from a different source. The reader or receiver can then carry out the experiment, and recover the FL-cDNA obtained. The product of the experiment reaction can be applied on an electrophoresis gel, according to known technique (example, Sambrook et al., 1989) and the FL-cDNA DNA band and the bands of the exons can be identified on the gel.
Accordingly, the present invention therefore provides a method for delivering and/or storing a printed material and/or support comprising the step of applying at least one set of primers on a printed material and/or support, the at least set of primers being capable of synthesizing FL-cDNA from the genomic DNA, and delivering and/or storing the printed material and/or support.
A doctor, who wishes to analyse one or more particular genes of a patient may obtains a blood or other biological material sample (template) from a patient. Then, he can use the kit according to the invention comprising a support comprising at least one or more sets of primers applied thereon, each set of primers specific for amplification and ligation of a specific FL-cDNA gene. Preferably, the support further comprises the enzyme, for instance Taq Polymerase, and buffer solution. The doctor or an assistant may recover the set of primers and optionally enzyme and buffer from the support and mix them with the template DNA. Carrying out the amplification (ex. PCR) process and electrophoresis. The electrophoresis shows the FL-cDNA gene. The doctor may immediately make a diagnosis in case of deletion/insertion of the particular gene.
The FL-cDNA obtained can also used for SNP analysis (for example sequencing) or for protein expression assay.
The present invention therefore provides for a Kit and/or a diagnostic kit comprising a support comprising at least one set of primers for the synthesis of cDNA and/or FL-cDNA from a template DNA. The present invention also provides for a diagnotic method comprising the steps of 1) preparing a template DNA (blood, fluid or other biological material) from a patient, 2) recovering at least the set of primers from the support, and optionally also recovering enzyme and buffer from the support, 3) mixing the template, set of primers, enzyme and buffer, 4) carrying out amplification and/or ligation process, 5) electrophoresis and 6) determining the diagnosis on the basis of the cDNA (exon) and/or FL-cDNA obtained.
The present invention also provides a method for preparing cDNA and/or FL-cDNA from a template comprising the steps of: 1) recovering at least one set of primers and optionally enzyme and buffer from a support, 2) mixing the set of primers, enzyme and buffer with the template, 3) carrying out the amplification and/or ligation of the exons, 4) electrophoresis, 5) optionally recovering of the cDNA and/or FL-cDNA from the electrophoresis means.
Further, the above method comprises the step of analysing the obtained cDNA and/or FL-cDNA for SNP, deletion or insertion analysis or the step of expressing a peptide, polynucleotide or protein. SNP and aberration analysis can be carried out by sequencing the cDNA and/or FL-cDNA or other known technique.
The peptide, polynucleotide or protein expression can be carried out by any peptide, polynucleotide or protein expression assay, for example the assay known as "Protein truncation test" or "Linked SP6/T7 in vitro transcription/translation kit" (2002 catalog number 188839 and 1814346, respectively), of Roche Diagnostic.
A method of preparation of full-length cDNA of hLH from genomic DNA according to the invention will be illustrated in more detail in example 8.
The support can also be in form of powder or solution preparation. Accordingly, the invention also provides a powder preparation comprising oligomer and/or polymer mixed with a carrier, for example methylcellulose. The carrier can be any inert carrier suitable to be mixed with the oligomer and/or polymer, for example any carrier usually utilized for the preparation of drugs. The powder preparation may further comprise enzyme and buffer solution.
The oligomer and/or polymer mixed with the carrier may be nucleic acid. Further, at least one primer or set of primers can also be mixed to the preparation. Optionally, enzyme and/or buffer can also mixed to the preparation.
The invention therefore provides a method for delivering and/storing a powder preparation as above comprising mixing a carrier to an oligomer and/or polymer, and delivering and/or storing such preparation. For example, the method comprises the steps of making a preparation comprising mixing nucleic acid and at least one primer or set of primers and optionally enzyme and buffer solution with an inert carrier, and delivering and/or storing such preparation. According to a particular realization, the nucleic acid is genomic DNA, and the at least one set of primers is capable of synthesizing full-length DNA from genomic DNA as above described. The invention therefore also relates to a method for preparing full-length DNA by comprising by using the preparation as above.
The preparation can also be in solution form. Accordingly, the oligomer and/or polymer may be mixed in a liquid carrier and included in water-soluble shell. Such water-soluble shell can be for example made is the same way of shell comprising drug according to known technique. The liquid preparation may contain nucleic acid and at least one primer or set of primers, and optionally buffer solution. Alternatively, the liquid preparation may comprise primer, enzyme and buffer but no nucleic acid. The liquid preparation can be dissolved into water solution with addition with the substance (for instance enzyme) necessary for starting the reaction.
EXAMPLES
The present invention will be explained in more detail with reference to the following examples. It should be noted, however, that the scope of the present invention is not limited by these examples.
Example 1
First, various types of sheets each having 5 mm.times.5 mm size (A) as well as having 10 mm.times.10 mm size (B) were prepared.
Two solutions were prepared as DNA samples. One solution (H solution) contained a plasmid DNA fragment of about 1.5 kbp including 1377 bp of .lambda.DNA fragment inserted in pBS at a site of EcORV at concentration of 333 ng/.mu.l. The other solution (F solution) contained the same plasmid DNA and fountain pen ink at concentrations of 333 ng/.mu.l and 17% (v/v) respectively.
Then, 3 .mu.l (1 .mu.g) each of the H solution and the F solution was spotted on a sheet having the size (A), while 6 .mu.l (2 .mu.g) each of the solutions was spotted on a sheet having the size (B), and these sheets were dried at 65.degree. C. for 30 minutes, respectively. Thereafter, the sheet having the size (A) was immersed into 200 .mu.l of water, while the sheet having the size (B) was immersed into 300 .mu.l of water, respectively. These immersed sheets were dried at 65.degree. C. for 10 minutes, and further they were treated at room temperature for 2 hours to thereby conduct elution.
Phenol extraction (extracting twice with phenol:chloroform:isoamyl alcohol=25:24:1) was repeated upon the eluate, and then DNA was recovered by an ethanol precipitation method.
The resulting DNA was dissolved in 10 .mu.l of water, and PCR was conducted as follows. After the first reaction at 94.degree. C. for 3 minutes, forty cycles of the reaction were repeated at 94.degree. C. for 1 minute and 68.degree. C. for 2 minutes with a final reaction volume: 25 .mu.l, a reaction composition: M13 (SEQ ID NO:1) primer (M3-30: 0.5 .mu.l of 10 .mu.M 5'-CAGTCACGACGTTGTAAAACGACGGCCAGT-3', 0.5 .mu.l of 10 .mu.M RV32 (SEQ ID NO:2): 5'-GATAACAATTTCACACAGGAAACAGCTATGAC-3'), 2.5 .mu.l of ExTaq 10.times. buffer solution, 2 .mu.l of 2.5 mM dNTP, 1 unit of ExTaq, and DNA. This DNA was a DNA template of about 1.5 kbp in size containing .lambda.DNA of 1377 bp in size together with plasmid DNA molecules located at both ends.
The base sequence of DNA (SEQ ID NO:3) was as follows:
1 GATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAT ATCGCATTTTTCACCATGCTCATCAAAGACAGTAAGATAAAACATTGTAA CAAAGGAATAGTCATTCCAACCATCTGCTCGTAGGAATGCCTTATTTTTT TCTACTGCAGGAATATACCCGCCTCTTTCAATAACACTAAACTCCAACAT ATAGTAACCCTTAATTTTATTAAAATAACCGCAATTTATTTGGCGGCAAC ACAGGATCTCTCTTTTAAGTTACTCTCTATTACATACGTTTTCCATCTAA AAATTAGTAGTATTGAACTTAACGGGGCATCGTATTGTAGTTTTCCATAT TTAGCTTTCTGCTTCCTTTTGGATAACCCACTGTTATTCATGTTGCATGG TGCACTGTTTATACCAACGATATAGTCTATTAATGCATATATAGTATCGC CGAACGATTAGCTCTTCAGGCTTCTGAAGAAGCGTTTCAAGTACTAATAA GCCGATAGATAGCCACGGACTTCGTAGCCATTTTTCATAAGTGTTAACTT CCGCTCCTCGCTCATAACAGACATTCACTACAGTTATGGCGGAAAGGTAT GCATGCTGGGTGTGGGGAAGTCGTGAAAGAAAAGAAGTCAGCTGCGTCGT TTGACATCACTGCTATCTTCTTACTGGTTATGCAGGTCGTAGTGGGTGGC ACACAAAGCTTTGCACTGGATTGCGAGGCTTTGTGCTTCTCTGGAGTGCG ACAGGTTTGATGACAAAAAATTAGCGCAAGAAGACAAAAATCACCTTGCG CTAATGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAG TGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCA AAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCA GCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTG TTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGATTTC AATTTTGTCCCACTCCCTGCCTCTGTCATCACGATACTGTGATGCCATGG TGTCCGACTTATGCCCGAGAAGATGTTGAGCAAACTTATCGCTTATCTGC TTCTCATAGAGTCTTGCAGACAAACTGCGCAACTCGTGAAAGGTAGGCGG ATCCCCTTCGAAGGAAAGACCTGATGCTTTTCGTGCGCGCATAAAATACC TTGATACTGTGCCGGATGAAAGCGGTTCGCGACGAGTAGATGCAATTATG GTTTCTCCGCCAAGAATCTCTTTGCATTTATCAAGTGTTTCCTTCATTGA TATTCCGAGAGCATCAATATGCAATGCTGTTGGGATGGCAATTTTTACGC CTGTTTTGCTTTGCTCGACATAAAGATATCAAGCTTGGCACTGGCCGTCG TTTTACAACGTCGTGACTG
After the reaction, 5 .mu.l of the reaction product was subjected to 1% agarose electrophoresis to detect (about 1.5 kbp of) a PCR product. The results are shown in FIG. 11.
Lane 1 is a DNA size marker (.lambda./StyI 200 .mu.g), lane 2 shows the fragment obtained by spotting the F solution onto a medical paper having the size (A), lane 3 shows the fragment obtained by spotting the F solution onto a medical paper having the size (B), lane 4 indicates the fragment obtained by spotting the F solution onto a copy paper having the size (A), lane 5 shows the fragment obtained by spotting the F solution onto a copy paper having the size (B), lane 6 shows the fragment obtained by spotting the H solution onto a medical paper having the size (A), lane 7 shows the fragment obtained by spotting the H solution onto a medical paper having the size (A), lane 8 shows the fragment obtained by spotting the H solution onto a copy paper having the size (A), lane 9 shows the fragment obtained by spotting the H solution onto a copy paper having the size (B), lane 10 is a fragment with no sheet (positive control), lane 11 is another fragment with no sheet (negative control), and lane 12 is a DNA size marker (.lambda./StyI 200 .mu.g).
Three .mu.l of DNA was applied to the lane 2, lane 3, and lane 6, respectively, while 1/50 .mu.l of the DNA was applied to the lane 4, lane 5, lane 7, lane 8, and lane 9, respectively.
More specifically, it is clear from the above described experimental results that the DNA fixed to the support in the DNA-fixed support prepared by the use of ordinary PPC or the like manufactured from cellulose as the support can be preserved at ordinary temperatures, and besides, the DNA fixed to the support can be recovered by elution from the support in the DNA-fixed support, and in addition, the DNA thus recovered by elution can be amplified by polymerase chain reaction.
In order to have the DNA adhere to the support, the following procedure may be adopted: DNA is picked up by use of a pin, the DNA on the pin is further transferred to the support, a DNA solution contained in a syringe is dropped onto the support so that the DNA adheres to it, and the DNA solution is allowed to adhere to the support in a printed state by utilizing an existing printing technique.
In this case, a printing technique such as an ink-jet printing system which is applied to ink-jet printers and the like may be utilized as the existing printing system.
In order to apply a printing technique such as an ink-jet printing system, the DNA solution is used in place of a coloring matter such as printing ink, and the support corresponding to printing paper may be printed by the use of the DNA solution in accordance with the ink-jet printing system.
Thus, the existing printing technique can be very easily applied to a method for supporting DNA-fixation according to the present invention because a DNA solution can be used in place of printing ink without any modification and it can be applied by an ink-jet printing system using a piezo-electric element or a heat-producing element.
In an ink-jet printing technique, dots around 20 .mu.m to 100 .mu.m in diameter can be usually printed, so it becomes possible to allow the DNA solution to adhere to a support at high density.
Moreover, DNA in a dried state is stable unlike other biomolecules such as protein, and it can withstand sufficiently a temperature of around 100.degree. C., so that an electronic printing or thermal transfer type printing technique including one is that actuated by a laser printer can also be applied to the method for supporting DNA-fixation according to the present invention.
Example 2
An article relating to the DNA molecule used in Example 1 was prepared which contained the title, the names of authors, abstract, introduction, materials and methods, results, discussion, acknowledgements and references. At the bottom of the article, letters "TEMPLATE" were printed using the F solution of Example 1 by an ink-jet printer (EPSON, PM-760C). As a result, clear printed letters were formed on the article.
Printing materials and supports according to the invention can be printed according to the techniques known in the art.
In case of scientific subjects, books, journals, magazines, papers, articles, supports and the like may be printed, for example, with letters and figures describing the research subject. Oligomers and/or polymers, for instance DNA solutions, are then applied, for instance spotted or printed, at defined or marked positions on the same sheet of paper or in a distinct sheet of paper or support of different size and shape. Optionally, an inert dye (for instance a red dye) can be added to the oligomer and/or polymer solution applied on the support so that the position of the spot can be visible on the support.
The sheets comprising the oligomers and/or polymers applied on them are bound in form of book, journal, or the like and delivered to readers through bookstores and by courier. Researchers, students and readers who have any interest in the enclosed genes can recover and use them immediately in their research.
In order to provide a successful printed material comprising at least one support having at least one oligomer and/or polymer applied thereon several issues must be considered. At first, the oligomer and/or polymer be easily extractable by readers or receivers, with an acceptably high success rate. Secondly, oligomer and/or polymer applied on the printed material or on the support should be preserved in a stable form during book binding and shipping. Thirdly, risk of contamination should be avoided.
The preparation of an efficient printed material and support according to the invention as well as the efficient preservation and recovery of oligomer and/or polymer has been investigated in the examples 3-7. In the following experiments we used use water-dissolvable paper to form the DNA sheet. We selected 60MDP paper (Mishima Paper Co., Ltd., Japan), which rapidly dissolves in water at room temperature.
Example 3
DNA Solution Preparation
We tested three RIKEN plasmid cDNA clones with various cDNA insert sizes (744 bp, 2440 bp and 5460 bp)(indicated as SEQ ID NOS: 4-6, respectively). The cDNAs were inserted into pBluescript according to known technique (Sambrook et al., 1989).
Plasmid DNA comprising the cDNA clones as above were purified using a Qiagen Spin Miniprep Kit (Qiagen, Japan) (alternatively, ultra-centrifugation methods, for example as described in Sambrook et al., 1989 can also be used). The plasmid DNA was dissolved in TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA. DNA concentration was adjusted to 0.1 .mu.g/.mu.l. At this step an inert dye, for example a red dye, can be added to the solution in order to facilitate identification of spot position on the support at the time of recovery. However, in the present experiment the plasmid DNA solution was spot on a marked place, so that the dye was not necessary.
Preparation of DNA Sheets
About 0.1 .mu.l of the plasmid DNA solution prepared as above was transferred onto 60MDP paper used as DNA sheet (Mishima Paper Co., Ltd, Japan) (the paper can be already printed or not) using a 96-pin tool (Multi 96-multiblot replicator VP409, Bio Medical Science Inc., US), which allowed us to spot defined amounts of DNA solution onto defined positions on the paper. Spotted positions were easily identified by being spotted in a marked position on the paper. (Alternatively, by the presence of red dye mixed into the DNA solution as discussed in the "DNA solution preparation" as above). We spotted the plasmid DNA solution five times for each spot, for a total of about 0.5 .mu.L of 0.05 .mu.g of plasmids.
Extraction and Recovery of DNA
After drying the paper in air for more than 30 minutes, we extracted DNA from the sheet as follows. The piece of 60MDP paper (0.4 mm.times.0.4 mm) containing the selected DNA spot was cut out from the sheet and placed into a PCR tube followed by addition of 50 .mu.L of PCR solution. PCR solution contained 1.5 U of KOD Plus DNA Polymerase (TOYOBO, Japan), 0.2 .mu.M of the following PCR primers: -21M13 (SEQ ID NO:7) 5'-TGTAAAACGACGGCCAGT-3' and 1233-Rv (SEQ ID NO:8): 5'-AGCGGATAACAATTTCACACAGGA-3'), 0.2 mM each of dATP, dGTP, dCTP and dTTP and in presence of various concentrations of MgCl.sub.2 (1 mM, 3.1 mM, 3.5 mM and 7.5 mM, respectively, as indicated in FIG. 12). After centrifuging the resulting solution, the PCR cycle was initiated. PCR cycles comprised 2 min at 94.degree. C.; 29 cycles of denaturing (94.degree. C., 1 min), annealing (55.degree. C., 1 min) and extension (75 sec at 68.degree. C.), and 15 min at 74.degree. C. Aliquots of PCR solutions were analyzed using 1% agarose gel electrophoresis carried out according to known technique (Sambrook et al., 1989).
As an alternative, an aliquot of the solution containing the dissolved DNA sheet, can undergo PCR in a separate tube, then Escherichia coli transformation, according to known technique (Sambrook et al., 1989). Readers or receiver can keep any remaining solution as backup or for other experiments.
Result: PCR Recovery of DNAs Spotted on the DNA Sheet
Purpose of Examples 4-7
DNA sheets and DNA books must be able to endure the various conditions of which they are subject throughout the book binding process by the manufacturer or by the publisher, shipment to readers or receivers and preservation of them in ordinary rooms. Temperature, pressure, humidity, light and physical rubbing represent the major problems with the potential to qualitatively affect the DNA sheets and books. These conditions have been tested and results are reported in the followings examples 4-7. The preparation of DNA and DNA sheets was carried out in the same way and using the same clones as example 3. In the experiments of example 4-7 and as shown in
Example 4
Preservation of DNA Sheets Under Temperature Conditions
DNA sheets were treated at 140.degree. C. for 30 seconds and at -40.degree. C. for 12 hours, respectively. All the cDNA inserts were successfully amplified with PCR and recovered. In
FIG. 13 and
This result confirms that the DNA applied on sheets and books is tolerant to high temperature that may be experienced during book binding process, and the low temperatures that may be experienced during air transport.
Example 5
Preservation of DNA sheets Under Pressure Conditions
DNA sheets were kept under high pressure conditions from about 90-170 Kgf/cm.sup.2 (pressure unit conversion is 10.2 kgf/cm.sup.2=1 Mpa).
A first purpose of the experiment was to check that DNA has not been transferred from paper 2 to paper 1 during the high pressure; this represent a contamination risk test. A further purpose of the experiment was to check that the cDNA clones applied on paper 2 was not damaged by the high pressure and can be successfully recovered and amplified.
Lanes 1, 2 and 3 are the control. They represent the clones 744 bp, 2440 bp and 5460 bp, respectively recovered from a DNA paper not subject to high pressure.
Lanes 4, 5 and 6 show the three cDNAs recovered from paper 2 subject to different values of high pressure. The pressure applied was 97.4 Kgf/cm.sup.2 for the clones of lanes 4 and 5 and 125.2 Kgf/cm.sup.2 for lane 5. This shows that the cDNAS of lanes 4, 5 and 6 were successfully amplified and recovered. Lanes 7, 8 and 9 refer to papers 1 subject to pressure in the same experiment of lanes 4, 5 and 6. No contamination can be observed in lanes 7, 8 and 9. This confirms that the cDNAs applied on papers 2 did not pass to paper 1 at these values of pressure.
Lanes 10, 11 and 12 relates to the three cDNAs recovered from paper2 underpressureof 148.4, 92.7 and 170.9 Kgf/cm.sup.2. All the cDNAs have been recovered under these pressure values. Lane 13, 14 and 15 refer to the papers 1 subject to the same experiments as lanes 10, 11 and 13. NO contamination was observed in lanes 13, 14 and 15.
This result confirms that the DNA applied on sheets and books is tolerant to high pressure that may be experienced during book binding process and no contamination of cDNA occurred under these conditions.
Example 6
Preservation of DNA Sheets Under Humidity Conditions
The preparation of DNA and DNA sheets was carried out in the same way and with the same clones as example 3.
DNA sheets were spotted with the three cDNA plasmids and kept in a humidified incubator at 37.degree. C. with 70% humidity for 12 hours. DNA inserts were then recovered and amplified by PCR as described above. We observed successful recovery of DNA from these DNA sheets.
Example 7
Preservation of DNA Sheets Under Rubbing Conditions
Furthermore, we tested whether rubbing of DNA sheets could cause any problems, such as preventing PCR amplification or introducing neighboring DNA. DNA sheets spotted with the same three cDNA plasmids were inserted into the book, and strongly shaken using a rotating shaker (180 rpm) at 37.degree. C. for 12 hours. DNA inserts were then recovered and amplified by PCR as described above.
As shown in
Example 8
Method for Preparing Full-Length cDNA from Genomic DNA
Human genomic DNA used as template DNA in this example was purchased from BD Biosciences Clontech, US.
Primers for synthesizing full length cDNA of human luteinizing hormone (hLH) were purchased from Invitrogen, US.
The full-length gene of human luteinizing hormone, which is the result of the present experiment, has is 503 bp and the following sequence (also reported as SEQ ID NO:9) (herebelow the sequence corresponding to exon 1 is written in capital letters and that of exon 2 in small letters): 1
The sequence of exon 1 is reported in SEQ ID NO:10. The sequence of exon 2 is reported in SEQ ID NO:11.
The sequences of the primers (the underline indicates overlapping region) were the following:
2 HsLH1F: CCAGGGGCTGCTGCTGTTG (SEG ID NO:12) HsLH1Rt: cagcacgcgcatCATGGTGGGGCAGTAGCC (SEG ID NO:13) HsLH2Ft: TGCCCCACCATGatgcgcgtgctgcaggcg (SEG ID NO:14) HsLH2R: tgcggattgagaagcctttattg (SEG ID NO:15)
HsLH1F and HsLH1Rt have complementary sequences to each end of exon 1 of human luteinizing hormone, and HsLH2F and HsLH2Rt have complementary seqnences to each end of exon 2 of the same gene. HsLH1Rt and HsLH2F have additional sequence complimentary to the next exon in order to ligate them to each other (FIG. 19)
The above primers were dissolved in 10 il of TE (10 mM Tris-HCl (pH8.0), 1 mM EDTA) with the final concentration of 10 pmol/il.
The primer solutions prepared as above was mixed for spot 1, 2, and 3.
Spot 1 solution: mixture of HsLH2F solution and HsLH2Rt solution with the ratio of 1:1;
Spot 2 solution: mixture of HsLH1F solution and HsLH1Rt solution with the ratio of 1:1;
Spot 3 solution: mixture of HsLH2F solution, HsLH2Rt solution, HsLH1F solution, and HsLH1Rt solution with the ratio of 1:1:1:1.
0.4 .mu.l of spot 1 solution, 0.4 .mu.l of spot 2 solution, and
0.8 .mu.l of spot 3 solution were transferred to each corresponding spot area on a 60MDP paper (Mishima Paper Co., Ltd, Japan) as shown in FIG. 20.
After drying the paper in air for more than 30 minutes, the primers were extracted from the sheet as follows. The pieces of 60MDP paper (0.4 mm.times.0.4 mm) containing the selected primer spot were cut out from the sheet and placed into three PCR tubes followed by addition of 50 .mu.l of PCR solution. PCR solution contained 10 mM Tris-HCl, pH8.3, 50 mM KCl, 2.5 mM MgCl.sub.2, 0.2 mM dNTP, 100 ng of template DNA (human genomic DNA, purchased from BD Biosciences Clontech, US), and 2.5 U of Taq DNA polymerase (Roche Diagnostics). PCR cycles comprised 3 min at 94.degree. C. (50 cycles: 94.degree. C. for 30 sec, 40.degree. C. for 30 sec, 72.degree. C. for 30 sec), and 72.degree. C. for 1 min.
5 il of each PCR final solution (PCR solution and primers) were analyzed using 3% NuSieve 3:1 agarose (TAKARA BIO INC., Japan) gel electrophoresis carried out according to known technique (Sambrook et al., 1989).
The entire disclosure of Japanese Patent Application No. 2001-291249 filed on Sep. 25, 2001 including specification, claims, drawings and summary is incorporated herein by reference in its entity.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entity.
INDUSTRIAL APPLICABILITY
In accordance with the present invention, printed materials comprising at least one support having at least one oligomer and/or polymer applied thereon are provided. Scientists can obtain oligomers and/or polymers of interest from the printed materials easily and immediately.
In accordance with the present invention, a method for delivering or storing at least one oligomer and/or polymer is provided. By this method, oligomers and/or polymers can be delivered and stored easily with reduced labor and time while eliminating the need to use special equipment or facilities.
Free Text of Sequence Listing
SEQ ID NO.1 shows the base sequence of M13 primer used in Example 1.
SEQ ID NO. 2 shows the base sequence of RV32 primer used in Example 1.
SEQ ID NO.3 shows the base sequence of the template DNA used in Example 1.
SEQ ID NOS: 4-6 show the sequences of the three cDNA mouse clones tested in Examples 3-7.
SEQ ID NO:7 shows the sequence of -21M13 primer used for the amplification of DNA of Example 3-7.
SEQ ID NO:8 shows the sequence of 1233-RV primer used for the amplification of DNA of Example 3-7.
SEQ ID NO: 9 is the sequence of the full-length human luteinizing hormone gene (cDNA) obtained in Example 8.
SEQ ID NOS: 10-11 show the sequences of exon 1 and exon 2, respectively, of the human luteinizing hormone (hLH).
SEQ ID NOS:12-15 shows the sequences of primers HSLH1F, HsLH1Rt, HsLH2Ft, HsLH2R for the amplification of the two exons of hLH and for the synthesis of hLH full-length.
Number | Date | Country | Kind |
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2001-291249 | Sep 2001 | JP | national |
Number | Date | Country | |
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0244623 A1 | Dec 2004 | US |