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US20030023077A1 - Method for extracting DNA from dried specimens - Google Patents

Method for extracting DNA from dried specimens Download PDF

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US20030023077A1
US20030023077A1 US10/123,532 US12353202A US2003023077A1 US 20030023077 A1 US20030023077 A1 US 20030023077A1 US 12353202 A US12353202 A US 12353202A US 2003023077 A1 US2003023077 A1 US 2003023077A1
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dna
formamide
pcr
tween
tris
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Thomas Scholl
Michael Pyne
Arnold Oliphant
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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  • the present invention relates to a composition and to a method for extracting DNA. More specifically, the present invention relates to a composition and to a method to extract DNA from dried biological samples on solid substrates, including but not limited to, buccal smears, semen and especially blood. The method can be conducted in a single-tube.
  • the DNA extracted in accordance with the present invention can be used for DNA amplification reactions, DNA sequencing, DNA restriction analysis and DNA hyridization.
  • PCR polymerase chain reaction
  • RFLP restriction fragment length polymorphism
  • Paper punchers are available, but this process is difficult to automate reliably. Static electricity and normal air movements can cause mishandling of the paper punches. Finally, the smallest available punchers are 1 mm in diameter, which does not fit in a 384-well microtiter plate. This places a lower limit on PCR reactions, which can increase the consumption of expensive reagents.
  • a DNA extraction composition (solution) which comprises formamide, citrate, a suitable buffer and optionally a non-ionic detergent.
  • a method for extracting DNA from biological samples is provided.
  • the biological sample is blood.
  • the blood is dried on a cellulosic material. The method comprises contacting the biological sample with the DNA extraction solution, heating the resultant mixture and isolating supernatant containing the extracted DNA. This method is suitable for extracting DNA in a single tube.
  • a composition which is capable of (i) extracting DNA from a biological sample, such as buccal smears, semen and particularly blood, dried on a cellulosic material, such as cotton based papers and (ii) removing or inactivating compounds present in the cellulosic material that may otherwise interfere in analysis of the DNA.
  • the DNA composition of the present invention is further capable of extracting sufficient DNA for molecular analysis in a single tube in a simple method.
  • the DNA extraction composition comprises (1) formamide, (2) citrate and (3) a buffer.
  • the DNA extraction composition may optionally comprises a non-ionic detergent.
  • Non-limiting examples of the buffer include acetate, BES, citrate, glycine, HEPES, MES, phosphate, PIPES, Tricine and Tris. It is preferred to use Tris.
  • the DNA extraction composition may optionally comprise a non-ionic detergent from about 0.1% to about 50%, preferably from about 0.5% to about 10%, and more preferably about 1%.
  • Non-limiting examples of non-ionic detergent include Nonidet NP-40, Triton® X-100 (octoxynol), Tween® 20 (polyoxyethylenesorbitan monolaurate) and Tween® 80 (polyoxyethylenesorbitan monooleate). It is preferred to use Tween® 80.
  • a simple, rapid, high-throughput method is provided to release DNA from a biological sample, especially from a biological sample adsorbed to a cellulosic material, such as cotton based papers.
  • the method can be performed in a single tube, thus greatly simplfying the DNA extraction process for biological samples, especially blood, dried on cotton based papers.
  • the DNA extraction method comprises (a) contacting a biological sample with the DNA extraction composition described above, (b) incubating the mixture at a low temperature, (c) incubating the mixture at an elevated temperature and (d) isolating the supernatant which contains the solubilized, extracted DNA.
  • a disk of cellulosic material containing a biological material is added to a tube or a well, such as a microtiter well. Any mass of substrate can be immersed in this composition for DNA extraction.
  • Commercial paper punches are available that produce sizes useful for 96-well format plates (1-5 mm).
  • the process of the present invention performs well with a variety of punch sizes and with various numbers of punches extracted as a single sample. These conditions include sizes of the disks is in the range from about 1 mm to about 5 mm, with between 1 and 20 punches per well.
  • the DNA extraction composition described herein is added to the tube or well containing the disk(s) in an amount from about 20 ⁇ L to about 300 ⁇ L, preferably from about 40 ⁇ L to about 100 ⁇ L, and more preferably about 50 ⁇ L.
  • the disk(s) and DNA extraction solution is first incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at a temperature of from about 4° C. to about 60° C., preferably from about 10° C. to about 45° C., and more preferably at room temperature (25° C.).
  • the disk(s) and DNA extraction solution is then incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at an elevated temperature of from about 45° C. to about 100° C., preferably from about 55° C. to about 100° C., and more preferably at 95° C.
  • the supernatant is then isolated using any suitable technique.
  • One suitable technique is centrifugation, such as 3000 ⁇ g for 10 minutes.
  • the supernatant can then be used in molecular techniques such as amplification reactions, hybridization analysis, sequencing and restriction analysis. It has been found that the supernatant can be used as target for amplification reactions following a dilution of 5-10 fold, preferably 8-fold, at a 10% to 20% final reaction volume.
  • the samples used in this procedure consisted either of EDTA-anticoagulated whole venous blood or capillary blood collected by standard procedures.
  • the blood was applied to either S&S 903 or FTATM blood collection cards and dried at room temperature. The dried cards were stored in the dark in plastic bags at room temperature.
  • PCR reactions were carried out in 384-well microtiter plates in 10 ⁇ L volumes using Amplitaq Gold (Perkin Elmer) and primers that amplify an approximately 560 bp region of Exon 11 of the BRCA1 tumor-suppressor gene. Targets are described below and also included positive and negative controls.
  • PCR products were electrophoresed on 1% SeaKem GTG (FMC) agarose with 100 bp ladder or ⁇ HindIII fragment size standards. The gels were stained with ethidium bromide and photographed using a Kodak MP4+ Polaroid camera system.
  • FMC SeaKem GTG
  • the compounds and concentrations tested in the PCR reactions are listed in Table 1. These PCR reactions used 20 ng genomic DNA as a target.
  • the FTA supernatant was prepared by incubating eight 3 mm bloodstained FTATM disks in 500 ⁇ L of water for 20 minutes, centrifuging briefly, and collecting the supernatant. FTA supernatant and SDS both proved to be potent inhibitors of PCR.
  • the candidate compounds for inhibition reversal have been titrated to determine upper thresholds for their concentrations in PCR reactions. Those concentrations, shown in Table 1, that do not adversely affect PCR were further tested for their ability to reverse PCR inhibition.
  • S&S 903 supernatant was prepared by incubating 8-3 mm bloodstained S&S 903 disks in 500 ⁇ L water for 5 minutes at 95° C., centrifuging briefly, and collecting the supernatant. This supernatant and SDS were tested as above for inhibition of PCR. Two-fold serial dilutions indicated that 0. 1% SDS completely inhibited PCR, while 50-12.5% S&S 903 supernatant partially inhibited PCR.
  • SDS concentrations of 0.02, 0.01 and 0.005% and S&S 903 supernatant concentrations of 25, 12.5, and 6.25% were chosen as the concentrations in PCR to test reversal of inhibition. These three concentrations of inhibitors were each tested against three concentrations of reagent that might reverse inhibition. Table 2 details the results of this experiment. All of the nonionic detergents were capable of reversing SDS inhibition to some extent. The S&S 903 supernatant seemed to extract insufficient material to inhibit PCR.
  • Amplification was detected in samples extracted with 100 ⁇ L 1:1 formamide solution:water for dilutions 2 ⁇ 4 through 2 ⁇ 7 and from those extracted with 500 ⁇ L water for dilutions 2 0 and 2 ⁇ 1 . This is the first successful amplification from DNA extracted by a one-step process.
  • the PCR reaction may be improved by identifying alternatives to formamide which solubilize material, but are less inhibitory, or using decreased concentrations of formamide.
  • citrate was tested as an inhibitor of DNA degradation during the heating process.
  • Eight 3 mm bloodstained S&S 903 disks were extracted in 150 ⁇ L of the solutions listed in table 7 using standard protocols. The supernatants were diluted by adding 450 ⁇ L water. A two-fold dilution series was used as a target (one-half the volume of the reaction) in standard PCR reactions. The reactions were qualitatively graded ( ⁇ poor to ++++ very good) for performance as listed in Table 7. Citrate performed very well in the extraction and PCR. However, all reactions that contained 5 mM citrate (the highest final concentration) were partially inhibited.
  • Disks were extracted in 10% formamide, 50 mM Tris pH 8.3, and 20 mM citrate as described in Table 8 using standard procedures. A two-fold dilution series was used as target (one-half the volume of the reaction) in standard PCR reactions.
  • the 6 mm disks were too big to be used in a 96-well plate format. They do not fall freely to the bottom of the well. Extraction of DNA from a single 3 mm or 3.2 mm disk produced less product than extractions with more or larger punches. A single 4.7 mm punch produced amplified product under all conditions tested. A single 4.7 mm punch extracted in approximately 50 ⁇ L is likely the best system.
  • a simple extraction technique for releasing DNA from bloodstained S&S 903 and FTATM cards has been described. This technique uses single paper disks and can be performed in a single tube with a simple extraction solution (10% formamide, 50 mM Tris pH 7.8, 20 mM citrate). Tween®80 at 1% concentration improves amplification of extracts from FTA paper. The system works well for PCR amplification and has the potential to be used for genotyping in multiplex reactions.

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Abstract

The present invention relates to a composition and to a method for extracting DNA. More specifically, the present invention relates to a composition and to a method to extract DNA from dried biological samples on solid substrates, including but not limited to, buccal smears, semen and especially blood. The method can be conducted in a single-tube. The DNA extracted in accordance with the present invention can be used for DNA amplification reactions, DNA sequencing, DNA restriction analysis and DNA hyridization.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of U.S. patent application Ser. No. 09/513,179 filed Feb. 25, 2000, which is related to U.S. Provisional Patent Application Serial No. 60/122,113 filed Feb. 26, 1999.[0001]
    • BACKGROUND OF THE INVENTION
  • The present invention relates to a composition and to a method for extracting DNA. More specifically, the present invention relates to a composition and to a method to extract DNA from dried biological samples on solid substrates, including but not limited to, buccal smears, semen and especially blood. The method can be conducted in a single-tube. The DNA extracted in accordance with the present invention can be used for DNA amplification reactions, DNA sequencing, DNA restriction analysis and DNA hyridization. [0002]
  • Blood and other biological samples are commonly archived by applying the sample to filter paper and allowing it to dry. These samples are used for newborn screening, diagnostic testing, and felon databasing. Samples are typically applied to filter paper cards and allowed to dry. Two types of filter paper cards are prevalent, Schleicher & Schuell 903 (S&S 903) and Fitzco FTA™ cards. S&S 903 paper is a heavy, highly absorbent cotton bond paper. Fitzco FTA™ cards are similar, but are treated with several compounds (U.S. Pat. No. 5,496,562) designed to kill pathogens and resist bacterial growth and DNA degradation. These compounds include Tris, EDTA, SDS, and uric acid. Fitzco claims that the “membranes are disrupted and the DNA explodes out of the nucleus causing high molecular weight DNA to become entangled in the fibers of the paper” (Fitzco Product Information). [0003]
  • To perform molecular diagnostic studies such as polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), the DNA must be purified in situ or extracted from the paper matrix. The currently available protocols require lengthy enzymatic digestions, incubations, and separation steps. Many of these protocols produce very little, poor quality DNA and are not amenable to high-throughput applications. Furthermore, protocols for FTA paper recommend amplification directly from the paper, since it is reported that it is difficult to get the DNA into solution. [0004]
  • There are several drawbacks to this last approach. There is no method to measure the amount or purity of DNA available in the paper. The protocol for RFLP requires enzymatic digestion to release the DNA from the paper matrix. Since the RFLP protocol involves enzymatic digestion, it poses the problems mentioned above. [0005]
  • Paper punchers are available, but this process is difficult to automate reliably. Static electricity and normal air movements can cause mishandling of the paper punches. Finally, the smallest available punchers are 1 mm in diameter, which does not fit in a 384-well microtiter plate. This places a lower limit on PCR reactions, which can increase the consumption of expensive reagents. [0006]
  • Therefore, it is desirable to develop a simple, rapid, high-throughput method to release the DNA from the paper matrix and a composition to be used in this method. DNA in solution is more easily applied to automated processes. [0007]
    • SUMMARY OF THE INVENTION
  • In accordance with one embodiment of the present invention, a DNA extraction composition (solution) is provided which comprises formamide, citrate, a suitable buffer and optionally a non-ionic detergent. In accordance with a second embodiment of the present invention, a method for extracting DNA from biological samples is provided. In one aspect of the invention, the biological sample is blood. In a second aspect of the invention, the blood is dried on a cellulosic material. The method comprises contacting the biological sample with the DNA extraction solution, heating the resultant mixture and isolating supernatant containing the extracted DNA. This method is suitable for extracting DNA in a single tube. [0008]
    • DETAILED DESCRIPTION OF THE INVENTION
  • Current procedures for extracting DNA from certain biological samples, such as dried blood on cellulosic material such as cotton based papers (e.g., Schleicher & Schuell 903 (S&S 903) and Fitzco FTA™ cards), require lengthy enzymatic digestions, incubations and separation steps. In addition, compounds used as preservatives in FTA™ paper would become soluble as a result of DNA extraction or DNA amplification reactions and inhibit enzymes used for DNA amplification. Thus, it was desired to develop a simple, rapid, high-throughput method to release the DNA from the paper matrix. It was further desired to develop a composition to be used in this method which would not only serve to extract the DNA, but would also serve to remove or inactivate the compounds present in FTA™ paper. The method and composition described herein satisfies these desires and produces DNA that is suitable for use in molecular procedures, including amplification, sequencing, hybridization and restriction analysis. [0009]
  • In accordance with one aspect of the present invention, a composition is provided which is capable of (i) extracting DNA from a biological sample, such as buccal smears, semen and particularly blood, dried on a cellulosic material, such as cotton based papers and (ii) removing or inactivating compounds present in the cellulosic material that may otherwise interfere in analysis of the DNA. The DNA composition of the present invention is further capable of extracting sufficient DNA for molecular analysis in a single tube in a simple method. According to the present invention, the DNA extraction composition comprises (1) formamide, (2) citrate and (3) a buffer. The DNA extraction composition may optionally comprises a non-ionic detergent. [0010]
  • The DNA composition comprises formamide in an amount from about 5% to about 90%, preferably from about 5% to about 50%, and more preferably about 10%. The DNA extraction composition comprises citrate in an amount from about 5 mM to about 60 mM, preferably from about 10 mM to about 40 mM, and more preferably about 20 mM. The DNA extraction composition comprises a buffer in the amount from about 1 mM to about 300 mM, preferably from about 10 mM to about 150 mM, more preferably about 50 mM. The pH of the buffer is from about 6.0 to about 8.8, preferably from about 7.5 to about 8.3, and more preferably about 7.8. Non-limiting examples of the buffer include acetate, BES, citrate, glycine, HEPES, MES, phosphate, PIPES, Tricine and Tris. It is preferred to use Tris. The DNA extraction composition may optionally comprise a non-ionic detergent from about 0.1% to about 50%, preferably from about 0.5% to about 10%, and more preferably about 1%. Non-limiting examples of non-ionic detergent include Nonidet NP-40, Triton® X-100 (octoxynol), Tween® 20 (polyoxyethylenesorbitan monolaurate) and Tween® 80 (polyoxyethylenesorbitan monooleate). It is preferred to use Tween® 80. [0011]
  • In accordance with a second aspect of the present invention, a simple, rapid, high-throughput method is provided to release DNA from a biological sample, especially from a biological sample adsorbed to a cellulosic material, such as cotton based papers. The method can be performed in a single tube, thus greatly simplfying the DNA extraction process for biological samples, especially blood, dried on cotton based papers. According to the present invention, the DNA extraction method comprises (a) contacting a biological sample with the DNA extraction composition described above, (b) incubating the mixture at a low temperature, (c) incubating the mixture at an elevated temperature and (d) isolating the supernatant which contains the solubilized, extracted DNA. [0012]
  • A disk of cellulosic material containing a biological material is added to a tube or a well, such as a microtiter well. Any mass of substrate can be immersed in this composition for DNA extraction. Commercial paper punches are available that produce sizes useful for 96-well format plates (1-5 mm). The process of the present invention performs well with a variety of punch sizes and with various numbers of punches extracted as a single sample. These conditions include sizes of the disks is in the range from about 1 mm to about 5 mm, with between 1 and 20 punches per well. The DNA extraction composition described herein is added to the tube or well containing the disk(s) in an amount from about 20 μL to about 300 μL, preferably from about 40 μL to about 100 μL, and more preferably about 50 μL. The disk(s) and DNA extraction solution is first incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at a temperature of from about 4° C. to about 60° C., preferably from about 10° C. to about 45° C., and more preferably at room temperature (25° C.). The disk(s) and DNA extraction solution is then incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at an elevated temperature of from about 45° C. to about 100° C., preferably from about 55° C. to about 100° C., and more preferably at 95° C. The supernatant is then isolated using any suitable technique. One suitable technique is centrifugation, such as 3000× g for 10 minutes. The supernatant can then be used in molecular techniques such as amplification reactions, hybridization analysis, sequencing and restriction analysis. It has been found that the supernatant can be used as target for amplification reactions following a dilution of 5-10 fold, preferably 8-fold, at a 10% to 20% final reaction volume.[0013]
    • EXAMPLES
  • The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized. [0014]
    • Example 1
  • Materials and Methods [0015]
  • Blood Samples and Paper Cards: [0016]
  • The samples used in this procedure consisted either of EDTA-anticoagulated whole venous blood or capillary blood collected by standard procedures. The blood was applied to either S&S 903 or FTA™ blood collection cards and dried at room temperature. The dried cards were stored in the dark in plastic bags at room temperature. [0017]
  • PCR Amplification: [0018]
  • PCR reactions were carried out in 384-well microtiter plates in 10 μL volumes using Amplitaq Gold (Perkin Elmer) and primers that amplify an approximately 560 bp region of Exon 11 of the BRCA1 tumor-suppressor gene. Targets are described below and also included positive and negative controls. [0019]
  • Agarose Gel Electrophoresis: [0020]
  • PCR products were electrophoresed on 1% SeaKem GTG (FMC) agarose with 100 bp ladder or λ HindIII fragment size standards. The gels were stained with ethidium bromide and photographed using a Kodak MP4+ Polaroid camera system. [0021]
    • Example 2
  • Identifying Reagent Candidates [0022]
  • Since components of blood (hemoglobin) and FTA™ paper (SDS) are known to inhibit PCR amplification, we sought to identify reagents that could reverse the inhibition of PCR by hemoglobin and SDS. First, we established concentrations of these compounds compatible with PCR amplification. [0023]
  • The compounds and concentrations tested in the PCR reactions are listed in Table 1. These PCR reactions used 20 ng genomic DNA as a target. The FTA supernatant was prepared by incubating eight 3 mm bloodstained FTA™ disks in 500 μL of water for 20 minutes, centrifuging briefly, and collecting the supernatant. FTA supernatant and SDS both proved to be potent inhibitors of PCR. The candidate compounds for inhibition reversal have been titrated to determine upper thresholds for their concentrations in PCR reactions. Those concentrations, shown in Table 1, that do not adversely affect PCR were further tested for their ability to reverse PCR inhibition. [0024]
    TABLE 1
    Titration of Compounds in PCR
    FTA Acetyl Form- Hist- PEG Tricine Triton ® Tween ® Tween ®
    Supernatant SDS Betaine BSA BSA DMSO EDTA amide Gelatin Glycerol idine MP-40 80000 pH 7.8 X-100 20 80
    % % mM mg/ml mg/ml % mM % mg/ml % mM % % mM % % %
    1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 50.0000 25.0000 12.5000 6.2500 3.1250 1.5625 0.7813 0.3906 0.19530.0977 0.0488 0.0244 0.0122 0.0061
    # 0.0031 5.00002.50001.25000.62500.31250.15630.07810.03910.01950.00980.00490.00240.00120.00060.0003 2.50001.2500 +UZ,14/17 0.6250
    Figure US20030023077A1-20030130-C00001
     0.0195 0.0098 0.0049 0.0024 0.0012 0.0006
    # 0.0003 0.0002 10.0000 5.0000 2.5000
    Figure US20030023077A1-20030130-C00002
     0.0781 0.0391 0.0195 0.0098 0.0049 0.0024 0.0012 0.0006    		 0.50000.25000.12500.06250.03130.01560.00780.0039 0.0020 +UZ,25/28 0.0010 0.0005 0.0002
    # 0.0001 0.0001 0.0000 50.000025.000012.50006.25003.1250 1.5625
    Figure US20030023077A1-20030130-C00003
     0.0488 0.0244 0.0122 0.0061 0.0031    		 20.000010.00005.00002.50001.2500
    Figure US20030023077A1-20030130-C00004
     0.0391 0.0195 0.0098 0.0049
    # 0.0024 0.0012 50.000025.000012.50006.25003.12501.56250.7813
    Figure US20030023077A1-20030130-C00005
     0.0244 0.0122 0.0061 0.0031    		 10.00005.00002.50001.25000.6250 0.3125 +UZ,45/48 0.1563
    Figure US20030023077A1-20030130-C00006
     0.0049 0.0024 0.0012
    # 0.0006 20.000010.0000 +UZ,51/54 5.0000
    Figure US20030023077A1-20030130-C00007
     0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 0.0024 0.0012    		 25.000012.50006.25003.12501.56250.78130.39060.19530.0977 0.0488 0.0244 0.0122 0.0061 0.0031 0.0015 20.0000 10.0000
    # 5.0000
    Figure US20030023077A1-20030130-C00008
     0.1563 0.0781 0.0391 0.0195 0.0098
    # 0.0049 0.0024 0.0012 20.000010.00005.0000 2.5000
    Figure US20030023077A1-20030130-C00009
     0.0781 0.0391 0.0195 0.0098 0.0049 0.0024 0.0012    		 250.0000125.000062.500031.250015.62507.8125 +UZ,71/74 3.90631.9531 0.9766
    # 0.4883 0.2441 0.1221 0.0610 0.0305 0.00153 10.0000 5.0000
    Figure US20030023077A1-20030130-C00010
     0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 0.0024 0.0012 0.0006    		 20.0000 10.0000 5.0000
    Figure US20030023077A1-20030130-C00011
     0.1563 0.0781 0.0391
    # 0.0195 0.0098 0.0049 0.0024 0.0012 10.0000 5.0000
    Figure US20030023077A1-20030130-C00012
     0.1563 0.0781 0.0391 0.0195 0.0098 0.0049 0.0024 0.0012 0.0006
    Key: No PCR product detected
    Reduced PCR product detected
    Figure US20030023077A1-20030130-C00013
    • Example 3
  • Testing PCR Inhibition by Hemoglobin and SDS [0025]
  • The purpose of the next experiment was to test PCR inhibition by hemoglobin and SDS separately. S&S 903 supernatant was prepared by incubating 8-3 mm bloodstained S&S 903 disks in 500 μL water for 5 minutes at 95° C., centrifuging briefly, and collecting the supernatant. This supernatant and SDS were tested as above for inhibition of PCR. Two-fold serial dilutions indicated that 0. 1% SDS completely inhibited PCR, while 50-12.5% S&S 903 supernatant partially inhibited PCR. SDS concentrations of 0.02, 0.01 and 0.005% and S&S 903 supernatant concentrations of 25, 12.5, and 6.25% were chosen as the concentrations in PCR to test reversal of inhibition. These three concentrations of inhibitors were each tested against three concentrations of reagent that might reverse inhibition. Table 2 details the results of this experiment. All of the nonionic detergents were capable of reversing SDS inhibition to some extent. The S&S 903 supernatant seemed to extract insufficient material to inhibit PCR. [0026]
    TABLE 2
    Reversal of PCR Inhibition
    903 Supernatant 25% 12.5% 6.25%
    Betaine (mM) 312.00 156.00 78.00 312.00 156.00 78.00 312.00 156.00 78.00
    BSA (mg/ml) 1.25 0.63 0.31 1.25 0.63 0.31 1.25 0.63 0.31
    Acetyl-BSA (mg/ml) 0.25 0.13 0.06 0.25 0.13 0.06 0.25 0.13 0.06
    DMSO (%) 5.00 2.50 1.25 5.00 2.50 1.25 5.00 2.50 1.25
    EDTA (mM) 0.63 0.31 0.16 0.63 0.31 0.16 0.63 0.31 0.16
    Formamide (%) 0.20 0.10 0.05 0.20 0.10 0.05 0.20 0.10 0.05
    Gelatin (%) 0.04 0.02 0.00 0.04 0.02 0.00 0.04 0.02 0.00
    Glycerol (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Histidine (mM) 0.25 0.13 0.06 0.25 0.13 0.06 0.25 0.13 0.06
    NP-40 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    PEG (%) 1.25 0.63 0.31 1.25 0.63 0.31 1.25 0.63 0.31
    Tricine (mM) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Triton ®X-100 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Tween ®20 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Tween ®80 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    SDS 0.02% 0.01% 0.005%
    Betaine (mM) 312.00 156.00 78.00 312.00 156.00 78.00 312.00 156.00 78.00
    BSA (mg/ml) 1.25 0.63 0.31 1.25 0.63 0.31 1.25 0.63 0.31
    Acetyl-BSA (mg/ml) 0.25 0.13 0.06 0.25 0.13 0.06 0.25 0.13 0.06
    DMSO (%) 5.00 2.50 1.25 5.00 2.50 1.25 5.00 2.50 1.25
    EDTA (mM) 0.63 0.31 0.16 0.63 0.31 0.16 0.63 0.31 0.16
    Formamide (%) 0.20 0.10 0.05 0.20 0.10 0.05 0.20 0.10 0.05
    Gelatin (%) 0.04 0.02 0.00 0.04 0.02 0.00 0.04 0.02 0.00
    Glycerol (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Histidine (mM) 0.25 0.13 0.06 0.25 0.13 0.06 0.25 0.13 0.06
    NP-40 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    PEG (%) 1.25 0.63 0.31 1.25 0.63 0.31 1.25 0.63 0.31
    Tricine (mM) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Triton ®X-100 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Tween ®20 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    Tween ®80 (%) 2.50 1.25 0.63 2.50 1.25 0.63 2.50 1.25 0.63
    • Example 4
  • Increasing Solubility of Extracted Material [0027]
  • In an attempt to increase the amount of material extracted from S&S 903 paper, the incubation time was increased and formamide was included in the composition. The objective was to test the ability of a formamide solution could extract more material from the blood spots and increase its solubility. Eight 3 mm bloodstained S&S 903 disks in 500 μL of water or 95% formamide in 100 mM Tris pH 8.3 at room temperature for 10 minutes. The disks were then incubated for 10 minutes at 95° C. The solutions were centrifuged and the supernatants collected. While the samples extracted with water produced large pellets of insoluble material, the samples extracted with formamide remained soluble. [0028]
  • These supernatants were tested for inhibition of PCR and for the presence of DNA. Each supernatant was tested in a two-fold dilution series starting at 25% supernatant in the standard PCR reaction. Each supernatant was also tested for DNA in a two-fold dilution series starting at 20% concentration as a target in PCR. The results are shown in Table 3. [0029]
  • The longer incubation seems to extract more material from the disks. The extraction with formamide produced almost no insoluble material and was a better target for PCR. Formamide extraction looks promising, although it is inhibitory at higher concentrations. [0030]
    TABLE 3
    Formamide Versus Water Extraction
    Inhibition Test for DNA
    Formamide % Water % Formamide % Water %
    25.0000 25.0000 20.0000 20.0000
    12.5000 12.5000 10.0000 10.0000
    6.2500 6.2500 5.0000  5.0000
    3.1250 3.1250 2.5000  2.5000
    1.5625 1.5625 1.2500  1.2500
    0.7813 0.7813 0.6250  0.6250
    0.3906 0.3906 0.3125  0.3125
    0.1953 0.1953 0.1563  0.1563
    0.0977 0.0977 0.0781  0.0781
    0.0488 0.0488 0.0391  0.0391
    0.0244 0.0244 0.0195  0.0195
    0.0122 0.0122 0.0098  0.0098
    0.0061 0.0061 0.0049  0.0049
    0.0031 0.0031 0.0024  0.0024
    0.0015 0.0015 0.0012  0.0012
    • Example 5
  • Optimizing Formamide Concentration in Extraction [0031]
  • Next, extractions were performed in reduced volumes and with reduced concentrations of formamide in an attempt to improve the formamide to DNA ratio while keeping the extracted material soluble. Eight 3 mm bloodstained S&S 903 disks were extracted in 500 or 100 μL volumes of 95% formamide in 100 mM Tris pH 8.3, water, or a 1:1 mixture of water and the 95% formamide/100 mM Tris solution as described earlier. These solutions were used in two-fold dilution series (2[0032] 0 through 2−12) as targets in PCR (2 μL in 10 μL reaction). Genomic DNA and no target controls were also performed. Amplification was detected in samples extracted with 100 μL 1:1 formamide solution:water for dilutions 2−4 through 2−7 and from those extracted with 500 μL water for dilutions 20 and 2−1. This is the first successful amplification from DNA extracted by a one-step process.
    • Example 6
  • Testing Alternatives to Formamide [0033]
  • The PCR reaction may be improved by identifying alternatives to formamide which solubilize material, but are less inhibitory, or using decreased concentrations of formamide. [0034]
  • The inhibitory effects of twelve reagents as alternatives to formamide were tested in PCR. The amplification was scored for performance by (1) failure-no detectable PCR product, (2) compromised-less PCR product compared to controls, or (3) nominal-PCR product similar to controls. The reagents and the concentrations in PCR that fit these scores are listed in Table 4. Overall, the glycols were less inhibitory to PCR than the amines. Inhibition by the amines was similar to that of formamide. Glycols can now be tested in the DNA extraction solution. [0035]
    TABLE 4
    Testing Alternatives to Formamide
    PCR Performance
    (reagent % in PCR)
    Reagent Failed Compromised Nominal
    4-Acetylmorpholine 10 5 2.5
    1,4-Butanediol 10 — 5
    Diethylene Glycol 20 — 10
    1,3-Dimethyl-s- 5 — 2.5
    Imidazolidinone
    Dipropylene Glycol 20 10  5
    3-Hydroxypropionitrile 10 — 5
    1-Methyl-2-Pyrrolidinone 10 5 2.5
    1,3-Propanedione 20 — 10
    2-Pyrrolidinone 10 5 2.5
    Tetramethylene Sulfone 10 5 2.5
    Triethylene Clycol 10 — 5
    Valerolactam 5   2.5 1.25
    • Example 7
  • Testing Glycols and Tween® 80 in Extraction [0036]
  • In previous experiments, DNA extracted from bloodstained cards with 50% formamide was successfully amplified. Also, Tween® 80, diethylene glycol, and 1,3 propanediol showed encouraging results. In this experiment, eight 3 mm bloodstained S&S 903 disks were extracted in 100 μL of solution (Table 5) using standard protocols. A two-fold dilution series was used in a standard PCR reaction. [0037]
  • All reactions with the glycols failed. The buffered formamide solutions results are similar to previous results, although the solution containing 5.94% formamide produced results that were not consistent. The solutions containing 10% or 5% Tween® 80 produced good results. [0038]
    TABLE 5
    Testing Tween ® 80 and Glycols in Extraction
    Detectable PCR
    Solution Dilution Range
      95% Formamide, 100 mM Tris pH 8.3 2−4-2−8
     47.5% Formamide, 100 mM Tris pH 8.3 2−2-2−11
    23.75% Formamide, 100 mM Tris pH 8.3 All failed
    11.88% Formamide, 100 mM Tris pH 8.3 2−1-2−2
     5.94% Formamide, 100 mM Tris pH 8.3 2−1-2−5
      20% Tween ® 80 All failed
      10% Tween ® 80 2−1-2−5
       5% Tween ® 80 2−1-2−7
      40% Diethylene Glycol All failed
      20% Diethylene Glycol All failed
      40% 1,3 Propanediol All failed
      20% 1,3 Propanediol All failed
    • Example 8
  • Combining Formamide and Tween® 80 in Extraction [0039]
  • To extend and combine the results from the last experiment, extraction solutions were prepared as described in Table 6. The solutions were used to extract DNA from eight 3 mm S&S bloodstained disks using the standard protocol. These solutions and two-fold serial dilutions in water were used as targets in a standard PCR reaction. [0040]
  • The solutions that produced the best PCR targets over the dilution range were (1) 11.9% formamide, 50 mM Tris pH 8.3 and (2) 5.9% formamide, 50 mM Tris pH 8.3. The addition of Tween® 80 seemed to slightly decrease the effectiveness of the extraction solution. [0041]
    TABLE 6
    Formamide and Tween ® 80 Combinations
    Extraction Solution
      95% Formamide, 50 mM Tris pH 8.3
    47.5% Formamide, 50 mM Tris pH 8.3
    23.8% Formamide, 50 mM Tris pH 8.3
    11.9% Formamide, 50 mM Tris pH 8.3
     5.9% Formamide, 50 mM Tris pH 8.3
      20% Tween ® 80
      10% Tween ® 80
      5% Tween ® 80
      20% Tween ® 80, 50 mM Tris pH 8.3
      10% Tween ® 80, 50 mM Tris pH 8.3
      5% Tween ® 80, 50 mM Tris pH 8.3
    47.5% Formamide, 50 mM Tris pH 8.3, 10% Tween ® 80
    23.8% Formamide, 50 mM Tris pH 8.3, 10% Tween ® 80
    11.9% Formamide, 50 mM Tris pH 8.3, 10% Tween ® 80
     5.9% Formamide, 50 mM Tris pH 8.3, 10% Tween ® 80
    47.5% Formamide, 50 mM Tris pH 8.3, 5% Tween ® 80
    23.8% Formamide, 50 mM Tris pH 8.3, 5% Tween ® 80
    11.9% Formamide, 50 mM Tris pH 8.3, 5% Tween ® 80
     5.9% Formamide, 50 mM Tris pH 8.3, 5% Tween ® 80
     2.5% Chelex, 10% Tween ® 80
      10% Tween ® 80, 1 mM EDTA
    • Example 9
  • Testing Citrate as Inhibitor of DNA Degradation During Heating [0042]
  • In an attempt to improve the extraction solution, citrate was tested as an inhibitor of DNA degradation during the heating process. Eight 3 mm bloodstained S&S 903 disks were extracted in 150 μL of the solutions listed in table 7 using standard protocols. The supernatants were diluted by adding 450 μL water. A two-fold dilution series was used as a target (one-half the volume of the reaction) in standard PCR reactions. The reactions were qualitatively graded (− poor to ++++ very good) for performance as listed in Table 7. Citrate performed very well in the extraction and PCR. However, all reactions that contained 5 mM citrate (the highest final concentration) were partially inhibited. [0043]
    TABLE 7
    Citrate in Extraction Buffer
    PCR performance
    (Citrate concentration)
    Solution None 10 mM 20 mM 40 mM
    Water − − + ++
    50 mM Tris pH 8.3 + ++ +++ +++
    50 mM Tris pH 8.3, 6% formamide ++ +++ +++ ++++
    50 mM Tris pH 8.3, 12% formamide ++ +++ ++++ ++++
    • Example 10
  • Determining Optimum Number and Size of Disks and Volume of Extraction [0044]
  • The next evaluation was designed to determine the number and size of disks and the volume of extraction solution to use. Disks were extracted in 10% formamide, 50 mM Tris pH 8.3, and 20 mM citrate as described in Table 8 using standard procedures. A two-fold dilution series was used as target (one-half the volume of the reaction) in standard PCR reactions. [0045]
  • The 6 mm disks were too big to be used in a 96-well plate format. They do not fall freely to the bottom of the well. Extraction of DNA from a single 3 mm or 3.2 mm disk produced less product than extractions with more or larger punches. A single 4.7 mm punch produced amplified product under all conditions tested. A single 4.7 mm punch extracted in approximately 50 μL is likely the best system. [0046]
    TABLE 8
    Testing Disk Number,
    Size and Extraction Volume
    Disk Number and Size Extraction Volume
    8 × 3 mm 100
    4 × 3 mm 100
    2 × 3 mm 100
    1 × 3 mm 100
    1 × 3.2 mm 100
    2 × 3.2 mm 100
    1 × 4.7 mm 100
    2 × 4.7 mm 100
    1 × 6 mm 100
    2 × 6 mm 100
    1 × 3.2 mm 80
    1 × 3.2 mm 60
    1 × 3.2 mm 40
    1 × 3.2 mm 20
    1 × 4.7 mm 80
    1 × 4.7 mm 60
    1 × 4.7 mm 40
    1 × 4.7 mm 20
    • Example 11
  • Testing Extraction on FTA™ Paper with Tween®80 [0047]
  • The next experiment was designed to test the optimized extraction buffer system on FTA paper. Since Tween®80 had previously been shown to reverse PCR inhibition due to SDS, a component of FTA™ paper, it was included in some PCR reactions. Bloodstained disks (one 4.7 mm S&S 903 or FTA™) were extracted in 50 μL of 50 mM Tris pH 8.3, 20 mM citrate, and 10% formamide using standard protocols. Then the supernatants were diluted three-fold in water, mixed, and centrifuged for 10 minutes at 3850 rpm. These solutions are defined as neat. The results of this experiment are shown in Table 9. These supernatants were diluted two-fold in water. This supernatant was serially diluted with water or Tween®80 solution and these solutions served as targets (5 μL in 10 μL reaction) in standard PCR. [0048]
  • This experiment produced good results with FTA™ paper provided that the PCR reaction included Tween®80. The inhibitors from the FTA™ paper do not completely inhibit PCR in the absence of Tween®80 with the target dilution of 2[0049] −4. Tween®80 at 1% produced better amplification than at a concentration of 0.2%. However, there might be some inhibition of amplification in extracts from S&S 903 paper at the higher Tween®80 concentration.
    TABLE 9
    Tween ® 80 in Extraction Solution
    PCR Result at Final Target Dilution
    Final Tween ® 80% 2−2 2−3 2−4
    S&S 903
    0.0 ++ ++ ++
    0.2 NA ++ ++
    1.0 NA ++ ++
    FTA ™
    0.0 − − +
    0.2 NA + +
    1.0 NA ++ ++
    • Example 12
  • Testing Effect of pH on Extraction Efficiency [0050]
  • The next experiment was designed to test the effect of varied pH on the extraction process. Bloodstained S&S 903 disks (one 4.7 mm) were extracted in 50 μL of 30 mM Tris (combinations of Tris base and Tris HCl indicated in Table 10), 20 mM citrate, and 10% formamide. The disks were incubated at room temperature for 10 minutes with agitation, incubated at 95° C. for 10 minutes, and diluted three-fold to a final Tris concentration of 20 mM. This dilution either normalized the Tris to a mixture of 50:50 Tris base:Tris HCl (normalized) or maintained the ratio in the extraction buffer (not normalized). Following dilution, the plates were centrifuged for 10 minutes at 3850 rpm and the supernatants were collected. All extractions and reactions were performed in duplicate. Two-fold serial dilutions of the solutions served as targets for amplification. [0051]
  • The extraction buffer functions well over a wide pH range. All dilutions amplified except those at 2[0052] −1 for 100% Tris HCl. Extractions at 20-40% Tris base generated the best amplification, although in 20% Tris base, the amplification was slightly weaker. Little difference was observed between amplifications from extracts that were normalized versus those that were not normalized. It is clear that pH below 8.3 has advantages. The PCR buffer used in these experiments has a pH between 7.8 and 7.9. This pH of extraction solution produced the best results.
    TABLE 10
    Test Extraction Buffer pH
    % Tris Base pH of Solution
    100 10.27
    80 8.73
    60 8.31
    50 8.11
    40 7.94
    20 7.51
    0 4.62
    • Example 13
  • Testing the Extraction System in Multiplex PCR [0053]
  • As a final test of the extraction buffer, a multiplex PCR reaction was tested. DNA was extracted from one 4.7 mm disk (either FTA or S&S 903) in 50 μL of extraction buffer (10% formamide, 50 mM Tris pH 8.3, 20 mM citrate) using standard protocols. These solutions were used as targets in Profiler+ genotyping reactions (3 μL reactions with 1 μL target). The extracted DNA solutions were diluted in Tween®80 solutions to give a final concentration of 1/12 or 1/24 target and 0, 0.13 and 0.67% Tween®80 in the PCR reaction. The Perkin Elmer buffer or our production PCR buffer supplemented with dNTPs at a final concentration in the reaction of 133 μM was used. [0054]
  • Only the control samples amplified in the Perkin Elmer buffer. The FTA and S&S 903 samples all failed. Surprisingly, all samples in production buffer amplified well. This is likely due to the pH resulting from combinations of PCR buffer and extraction buffer. Production PCR buffer seems more compatible than Perkin Elmer buffer using these extracts as a target. The more dilute samples (1/24) produced markedly lower signal. [0055]
    • Example 14
  • Testing the Extraction System with Alternative Dried Specimens [0056]
  • Replicate samples of dried buccal smears and semen were extracted in accordance with the above process. PCR amplification was performed on the DNA and the results analyzed. It was found that the PCR amplification was also successful for dried buccal smears and semen. [0057]
  • Conclusions: [0058]
  • A simple extraction technique for releasing DNA from bloodstained S&S 903 and FTA™ cards has been described. This technique uses single paper disks and can be performed in a single tube with a simple extraction solution (10% formamide, 50 mM Tris pH 7.8, 20 mM citrate). Tween®80 at 1% concentration improves amplification of extracts from FTA paper. The system works well for PCR amplification and has the potential to be used for genotyping in multiplex reactions. [0059]
  • While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. [0060]

Claims (14)

What is claimed is:
1. A composition for extracting DNA from a biological sample which comprises:
(a) 5% to 90% formamide;
(b) 5 mM to 60 mM citrate; and
(c) 1 mM to 300 mM buffer, pH 6.0 to 8.8, wherein the buffer is not phosphate.
2. The composition of claim 1 which further comprises 0.1% to 50% of a non-ionic detergent.
3. The composition of claim 1, wherein the formamide is present at 5% to 50%.
4. The composition of claim 1, wherein the citrate is present at 10 mM to 40 mM.
5. The composition of claim 1, wherein the buffer is present at 10 mM to 150 mM, pH 7.5 to 8.3.
6. The composition of claim 1 which comprises:
(a) 10% formamide;
(b) 20 mM citrate; and
(c) 50 mM buffer, pH 7.8.
7. The composition of claim 6, wherein said buffer is Tris.
8. The composition of claim 2, wherein the formamide is present at 5% to 50%.
9. The composition of claim 2, wherein the citrate is present at 10 mM to 40 mM.
10. The composition of claim 2, wherein the buffer is present at 10 mM to 150 mM, pH 7.5 to 8.3.
11. The composition of claim 2, wherein the non-ionic detergent is present at 0.5% to 10%.
12. The composition of claim 2 which comprises:
(a) 10% formamide;
(b) 20 mM citrate;
(c) 50 mM buffer, pH 7.8;
(d) 1% non-ionic detergent.
13. The composition of claim 12, wherein the buffer is Tris.
14. The composition of claim 12, wherein the non-ionic detergent is Tween 80.
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Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050287583A1 (en) * 1997-01-21 2005-12-29 Promega Corporation Methods and kits for isolating biological target materials using silica magnetic particles
US6027945A (en) * 1997-01-21 2000-02-22 Promega Corporation Methods of isolating biological target materials using silica magnetic particles
ES2188384B1 (en) * 2001-06-21 2005-01-01 Clinica Perio, S.L. PROCEDURE FOR OBTAINING HUMAN DNA FROM GINGIVAL CREVICULAR FLUID.
US7772383B2 (en) * 2002-01-25 2010-08-10 The Trustees Of Princeton University Chemical PCR: Compositions for enhancing polynucleotide amplification reactions
US7601491B2 (en) * 2003-02-06 2009-10-13 Becton, Dickinson And Company Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
EP1641944B1 (en) * 2003-07-09 2011-06-29 Genvault Corporation Room temperature elution of nucleic acids
US20050176027A1 (en) * 2003-10-14 2005-08-11 Suzow Joseph G. Nucleic acid extraction method
SI1871912T1 (en) * 2005-04-15 2012-06-29 Epigenomics Ag Method for determining DNA methylation in blood or urine samples
KR100710418B1 (en) 2006-02-09 2007-04-24 단국대학교 산학협력단 Die extract strip and die extract method comprising the same
US20080124777A1 (en) * 2006-11-29 2008-05-29 Canon U.S. Life Sciences, Inc. Method for releasing genetic material from solid phase
ES2532632T3 (en) * 2008-06-30 2015-03-30 Life Technologies Corporation Direct amplification method from raw nucleic acid samples
US8222397B2 (en) * 2009-08-28 2012-07-17 Promega Corporation Methods of optimal purification of nucleic acids and kit for use in performing such methods
US8039613B2 (en) 2009-08-28 2011-10-18 Promega Corporation Methods of purifying a nucleic acid and formulation and kit for use in performing such methods
WO2012083175A1 (en) 2010-12-17 2012-06-21 Wisconsin Alumni Research Foundation One-step method of elution of dna from blood samples
US20130157253A1 (en) * 2011-12-16 2013-06-20 Mei Wang Baker Detection of cytomegalovirus dna using amplification from blood samples
WO2017070643A1 (en) * 2015-10-22 2017-04-27 University Of Maryland, Baltimore Methods for extracting nucleic acids from dried blood and uses thereof
CN108977438A (en) * 2018-08-29 2018-12-11 武汉纳磁生物科技有限公司 A kind of extracting method and kit of partner DNA

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4752565A (en) * 1986-04-07 1988-06-21 The United States Of America As Represented By The Department Of Health And Human Services Cell line producing AIDS viral antigens without producing infectious virus particles
US4762780A (en) * 1984-04-17 1988-08-09 The Regents Of The University Of California Method and composition for screening and diagnosing "HCMV"
US5231015A (en) * 1989-10-18 1993-07-27 Eastman Kodak Company Methods of extracting nucleic acids and pcr amplification without using a proteolytic enzyme
US5234809A (en) * 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
US5334499A (en) * 1989-04-17 1994-08-02 Eastman Kodak Company Methods of extracting, amplifying and detecting a nucleic acid from whole blood or PBMC fraction
US5407823A (en) * 1990-04-06 1995-04-18 Institut National De La Sante Et De La Recherche Medicale Polypeptides having a dopaminergic receptor activity, nucleic acids coding for these polypeptides and use of these polypeptides for the screening of substances active on these polypeptides
US5596092A (en) * 1990-02-14 1997-01-21 Talent S.R.L. Extraction of genomic DNA from blood using cationic detergents
US5631132A (en) * 1993-05-20 1997-05-20 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acid probes and methods for detecting Candida glabrata DNA in blood
US5830759A (en) * 1994-08-18 1998-11-03 The Trustees Of Columbia University In The City Of New York Unique associated Kaposi's sarcoma virus sequences and uses thereof
US5861253A (en) * 1992-07-17 1999-01-19 Aprogenex, Inc. Intracellular antigens for identifying fetal cells in maternal blood
US5977316A (en) * 1995-01-17 1999-11-02 The Board Of Trustees Of The University Of Kentucky Monoclonal antibody 1A7 and related polypeptides
US6297370B1 (en) * 1991-05-08 2001-10-02 Chiron Corporation HCV genomic sequences for diagnostics and therapeutics

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5558096A (en) * 1978-10-25 1980-04-30 Noda Sangyo Kagaku Kenkyusho Method of making novel recombined dna
US5348855A (en) * 1986-03-05 1994-09-20 Miles Inc. Assay for nucleic acid sequences in an unpurified sample
US5063162A (en) * 1987-04-24 1991-11-05 Hoffmann-La Roche Inc. Process for isolating nucleic acids utilizing protease digestion
US5807527A (en) * 1991-05-29 1998-09-15 Flinders Technologies Pty. Ltd. Solid medium and method for DNA storage
US5496562A (en) * 1988-10-05 1996-03-05 Flinders Technologies Pty Ltd Solid medium and method for DNA storage
US6043032A (en) * 1993-09-22 2000-03-28 Tosoh Corporation Method of extracting nucleic acids and method of detecting specified nucleic acid sequences
JPH11507544A (en) * 1995-06-08 1999-07-06 プロジェン インダストリーズ リミテッド Method and apparatus for extracting DNA
GB2388803B (en) * 2002-01-14 2005-11-02 Tcl Supplies Ltd Drill bit

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4762780A (en) * 1984-04-17 1988-08-09 The Regents Of The University Of California Method and composition for screening and diagnosing "HCMV"
US4752565A (en) * 1986-04-07 1988-06-21 The United States Of America As Represented By The Department Of Health And Human Services Cell line producing AIDS viral antigens without producing infectious virus particles
US5234809A (en) * 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
US5334499A (en) * 1989-04-17 1994-08-02 Eastman Kodak Company Methods of extracting, amplifying and detecting a nucleic acid from whole blood or PBMC fraction
US5231015A (en) * 1989-10-18 1993-07-27 Eastman Kodak Company Methods of extracting nucleic acids and pcr amplification without using a proteolytic enzyme
US5596092A (en) * 1990-02-14 1997-01-21 Talent S.R.L. Extraction of genomic DNA from blood using cationic detergents
US5407823A (en) * 1990-04-06 1995-04-18 Institut National De La Sante Et De La Recherche Medicale Polypeptides having a dopaminergic receptor activity, nucleic acids coding for these polypeptides and use of these polypeptides for the screening of substances active on these polypeptides
US6297370B1 (en) * 1991-05-08 2001-10-02 Chiron Corporation HCV genomic sequences for diagnostics and therapeutics
US5861253A (en) * 1992-07-17 1999-01-19 Aprogenex, Inc. Intracellular antigens for identifying fetal cells in maternal blood
US5631132A (en) * 1993-05-20 1997-05-20 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acid probes and methods for detecting Candida glabrata DNA in blood
US5830759A (en) * 1994-08-18 1998-11-03 The Trustees Of Columbia University In The City Of New York Unique associated Kaposi's sarcoma virus sequences and uses thereof
US5977316A (en) * 1995-01-17 1999-11-02 The Board Of Trustees Of The University Of Kentucky Monoclonal antibody 1A7 and related polypeptides

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