US20120152743A1 - Method for electroeluting genetic material from dried samples - Google Patents

Method for electroeluting genetic material from dried samples Download PDF

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US20120152743A1
US20120152743A1 US12/972,236 US97223610A US2012152743A1 US 20120152743 A1 US20120152743 A1 US 20120152743A1 US 97223610 A US97223610 A US 97223610A US 2012152743 A1 US2012152743 A1 US 2012152743A1
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solid medium
dna
genetic material
sample
fixed
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Erin Jean Finehout
John Richard Nelson
Patrick McCoy Spooner
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General Electric Co
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General Electric Co
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Priority to US12/972,236 priority Critical patent/US20120152743A1/en
Assigned to GENERAL ELECTRIC COMPANY reassignment GENERAL ELECTRIC COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FINEHOUT, ERIN JEAN, NELSON, JOHN RICHARD, SPOONER, PATRICK MCCOY
Priority to JP2013544728A priority patent/JP6118262B2/ja
Priority to PCT/US2011/064823 priority patent/WO2012082849A1/en
Priority to EP11848353.6A priority patent/EP2652175B1/en
Publication of US20120152743A1 publication Critical patent/US20120152743A1/en
Abandoned legal-status Critical Current

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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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

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  • the subject matter disclosed herein relates to a solid medium for use in the storage of genetic material from biological samples and more particularly to methods to electroelute genetic material from the biological samples directly from the solid medium.
  • Various facilities include storage systems for large collections of genetic material (e.g., DNA) collected from a wide range of sources (e.g., human blood, cell lines, etc.).
  • the genetic material is stored in a safe, convenient, and minimally labor intensive manner within these storage systems for later analysis.
  • solid media such as Whatman® FTA® substrates, are used to store genetic material from various biological samples, such as tissues or cells.
  • extracting genetic material from the solid media involves additional labor, sometimes intensive labor when dealing with numerous samples.
  • extraction of genetic material may include either using detergents or incubating the solid media at high temperatures. The detergent may interfere with analysis of the genetic material, thus additional labor is required to remove the detergent. Also, the higher temperatures may fragment the genetic material.
  • there is a need to reduce the work required to extract genetic material, while also minimizing the fragmentation of the genetic material.
  • a method for extracting genetic material from a biological sample stored on a solid medium includes obtaining the solid medium, wherein the biological sample is applied on the solid medium, and the solid medium includes chemicals that lyzed the biological sample and preserved the genetic material. The method also includes electroeluting the genetic material directly from the solid medium to a subsequent medium.
  • a method for extracting genetic material from a fixed tissue sample stored on a solid medium includes applying at least a portion of the fixed tissue sample to the solid medium, wherein the solid medium includes chemicals that can lyse the cells or preserve the genetic material. The method further includes electroeluting the genetic material directly from the solid medium to a subsequent medium.
  • a method for extracting DNA from a biological sample stored on a cellulose-based paper includes obtaining the cellulose-based paper, wherein the biological sample is applied on the cellulose-based paper, and the cellulose based paper includes chemicals that lyse the biological sample and preserve the DNA.
  • the method also includes electroeluting DNA directly from the cellulose-based paper into an electrophoresis gel.
  • FIG. 1 is a flow chart illustrating a method for extracting genetic material from a biological sample stored on a solid medium in accordance with aspects of the present disclosure
  • FIG. 2 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using pulsed-field gel electrophoresis in accordance with aspects of the present disclosure
  • FIG. 3 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using alkaline gel electrophoresis in accordance with aspects of the present disclosure
  • FIG. 4 is a flow chart illustrating a method for extracting genetic material from a fixed tissue sample stored on a solid medium in accordance with aspects of the present disclosure
  • FIG. 5 depicts a SYBR® Gold stained electrophoresis gel, using a vertical polyacrylamide gel electrophoresis (PAGE) system, of multiplex PCR amplification of DNA that has been extracted from a human prostate sample fixed in formalin and applied to Whatman® FTA® paper;
  • PAGE vertical polyacrylamide gel electrophoresis
  • FIG. 6 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using native gel electrophoresis in accordance with aspects of the present disclosure
  • FIG. 7 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using native gel electrophoresis in accordance with aspects of the present disclosure.
  • FIG. 8 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using native gel electrophoresis in accordance with aspects of the present disclosure.
  • embodiments of the invention include a method for extracting genetic material (e.g., DNA) directly from biological samples stored on a solid medium (e.g., chemically treated cellulose substrate) using electroelution.
  • the method includes obtaining the solid medium that includes a stored biological sample previously applied and dried on the solid medium.
  • the solid medium includes chemicals that lyse the biological sample and preserve the genetic material.
  • the method also includes electroeluting the genetic material directly from the solid medium to a subsequent medium after removing the chemicals from the solid medium.
  • the subsequent medium may include an electrophoresis gel, a solution, or a capture surface (e.g., a blotting membrane).
  • fixed samples of cells or tissues may be applied to the solid medium.
  • the fixed samples may be processed prior to or after application to the solid medium.
  • processing of the fixed samples may include rehydrating and/or lyzing the fixed sample. Whether fixed or not, the samples may be further processed subsequent to application to the solid medium.
  • repair of nicks and abasic sites within the genetic material may occur, while the genetic material is in a fixed position on the solid medium.
  • the methods above provide a single platform for lyzing a biological sample, extracting DNA from the biological sample, binding the DNA to a surface, washing the DNA, and eluting high molecular weight (e.g., at least 10 kilobases) as wells as less fragmented DNA.
  • the ability to directly electroelute the genetic material from the solid medium avoids the use of detergents normally used to extract DNA as well as the extra steps necessary to remove the detergent and make the DNA usable for subsequent analysis.
  • directly electroeluting the genetic material from the solid medium without using high temperatures reduces the fragmentation of DNA.
  • researchers have had to insert the solid medium on which the DNA is located directly into subsequent genetic analysis reactions (e.g. PCR) to analyze the DNA that is bound on the solid medium. It can be appreciated that elution of DNA from the solid medium allows for the evaluation of the genetic material in more than just one reaction. Overall, the disclosed embodiments reduce the work of the user in extracting the DNA from the solid medium while improving the quality and availability of the DNA.
  • Biological samples used in the embodiments below may include physiological/pathological body liquids (e.g., secretions, excretions, exudates, and transudates) or cell suspensions (e.g., blood, lymph, synovial fluid, semen, saliva containing buccal cells, skin scrapings, hair root cells, etc.), liquid extracts or homogenates of cell suspensions of humans and animals; physiological/pathological liquids or cell suspensions of plants; liquid products, extracts or suspensions of bacteria, fungi, plasmids, viruses, etc.; liquid products, extracts, or suspensions of parasites including helminths, protozoas, spirochetes, etc.; human or animal body tissues (e.g., bone, liver kidney, etc.); media from DNA or RNA synthesis; mixtures of chemically or biochemically synthesized DNA or RNA; and any other source in which DNA and/or RNA is or can be in a liquid medium.
  • physiological/pathological body liquids e.g
  • FIG. 1 is a flow chart illustrating an embodiment of a method 10 for extracting genetic material (e.g., DNA) from a biological sample (e.g., tissues, cells, viruses, bacteriophages, or any other sample containing nucleic acid) stored on a solid medium.
  • a biological sample e.g., tissues, cells, viruses, bacteriophages, or any other sample containing nucleic acid
  • Processing of the biological sample may occur prior to application of the biological sample to the solid medium.
  • biological samples including cells or tissues may be fixed (e.g., in formalin).
  • Processing of the fixed tissue or cells may include rehydrating the fixed cells or tissues, lyzing the cells or tissues (e.g., with protease), and/or reversing cross-linking between the genetic material (e.g., DNA) and proteins. In certain embodiments, some of the processing, such as reversing cross-linking, may occur subsequent to application of the fixed cells or tissues to the solid medium.
  • reversing cross-linking may occur subsequent to application of the fixed cells or tissues to the solid medium.
  • the solid medium includes a chemically treated absorbent cellulose-based material.
  • the solid medium may include Whatman® FTA® or FTA® Elute paper (GE Healthcare).
  • the solid medium includes chemicals that lyze the biological sample (e.g., tissues or cells) and/or preserve the genetic material on the solid medium.
  • the solid medium and composition of chemicals may be as described in greater detail in U.S. Pat. No. 5,976,572, entitled “Dry Solid Medium for Storage and Analysis of Genetic Material,” and U.S. Pat. No. 5,985,327, entitled “Solid Medium and Method for DNA Storage,” both of which are hereby incorporated by reference in their entirety for all purposes.
  • the solid medium may include a weak base (e.g., tris-hydroxymethyl methane (tris)), chelating agent (e.g., ethylene diamine tetracetic acid (EDTA)), anionic surfactant or detergent (e.g., sodium dodecyl sulphate (SDS)), and/or uric acid or urate salt.
  • a weak base e.g., tris-hydroxymethyl methane (tris)
  • chelating agent e.g., ethylene diamine tetracetic acid (EDTA)
  • anionic surfactant or detergent e.g., sodium dodecyl sulphate (SDS)
  • SDS sodium dodecyl sulphate
  • uric acid or urate salt e.g., sodium dodecyl sulphate
  • the chemicals lyse the biological sample and denature proteins.
  • the chemicals inactivate nucleases and pathogens to allow for the preservation and long term storage
  • a portion of the solid medium containing the sample is removed for electroeluting the genetic material (e.g., DNA) from this portion (block 18).
  • the entire sample may be used.
  • the portion of the solid medium containing the biological sample may be rinsed to remove the chemicals (block 20).
  • rinsing the portion of the sample-containing solid medium includes soaking the solid medium portion in a buffer solution or flowing buffer through or across the solid medium portion.
  • the buffer solution includes an alkaline buffer solution (e.g., pH 8.0) including at least tris and EDTA (e.g., T.E.).
  • the solid medium portion with the sample may be soaked one or more times for a fixed time (e.g., 5 minutes) in the alkaline buffer solution.
  • the buffer solution includes Whatman® FTA® purification reagent from GE Healthcare.
  • the solid medium portion with the sample may be soaked one or more times for a fixed time (e.g., 5 minutes) in the FTA® purification reagent.
  • both the alkaline buffer solution and the FTA® purification reagent may be used to rinse the sample-containing solid medium portion.
  • the sample-containing solid medium portion may be soaked for a fixed time (e.g., 5 minutes each soaking) twice in the alkaline buffer solution and for a fixed time (e.g., 5 minutes each soaking) twice in the FTA® purification reagent.
  • rinsing the portion of the sample-containing solid medium includes soaking the solid medium portion in water or flowing water through or across the solid medium portion.
  • the solid medium portion with the sample may be soaked one or more times for a fixed time (e.g., 5 minutes) in the water.
  • electroelution e.g., electroelution in dialysis tubing
  • rinsing of the sample-containing solid medium may not be necessary.
  • the solid medium portion with the sample may be treated with enzymes (block 22) to reduce or eliminate non-DNA contaminants (e.g., proteins, lipids, carbohydrates, etc.).
  • enzymes e.g., proteins, lipids, carbohydrates, etc.
  • an enzymatic solution may include hydrolytic enzymes such as proteases, lipases, and/or glycoside hydrolases.
  • the genetic material e.g., DNA
  • the genetic material stored on the solid medium may include nicks (i.e., absence of phosphodiester bond between adjacent nucleotides) or abasic sites (also called AP sites, i.e., absence of a purine or pyrimidine base in a nucleotide while retaining the integrity of the phosphodiester ribose backbone) introduced via a fixing agent (e.g., formalin) in pre-processed samples or via other means.
  • nicks i.e., absence of phosphodiester bond between adjacent nucleotides
  • abasic sites also called AP sites, i.e., absence of a purine or pyrimidine base in a nucleotide while retaining the integrity of the phosphodiester ribose backbone
  • a fixing agent e.g., formalin
  • repairing the genetic material includes applying a solution containing at least DNA polymerase (e.g., E. coli DNA polymerase I) and DNA ligase (e.g., T4 DNA ligase or a bacterial DNA ligase) to repair the nicked damage.
  • DNA polymerase e.g., E. coli DNA polymerase I
  • DNA ligase e.g., T4 DNA ligase or a bacterial DNA ligase
  • the DNA polymerase includes DNA polymerase activity in addition to both 3′ to 5′ exonuclease activity to mediate proofreading and 5′ to 3′ exonuclease activity to mediate nick translation during DNA repair.
  • DNA polymerase requires dNTP's.
  • the DNA ligase includes enzymatic activity to form a phosphodiester bond between adjacent nucleotides.
  • DNA ligase requires either rATP or NAD.
  • the sample-containing solid medium portion may be incubated with the DNA repair solution at 37° C. for 30 minutes, and then incubated at 65° C. for 20 minutes.
  • the DNA repair solution may include other DNA repair enzymes with similar or different DNA repair mechanisms.
  • AP endonuclease e.g., E. coli endonuclease IV
  • the abasic site can be cleaved by an AP endonuclease, leaving 3 ′ hydroxyl and 5′ deoxyribosephosphate termini. This structure can be subsequently repaired by the combined action of DNA polymerase and DNA ligase.
  • the genetic material is directly electroeluted from the solid medium to a subsequent medium (block 26).
  • the sample-containing solid medium portion may be disposed directly into a well of an electrophoresis gel (e.g., agarose gel), the well sealed with agarose, and the genetic material directly electroeluted into the electrophoresis gel ( FIGS. 2 and 3 ).
  • the genetic material may be electroeluted into a solution. Electroelution into a solution may occur in a variety of ways.
  • D-tubeTM dialyzers (Merck Biosciences Ltd.), dialysis cassettes or tubing, Whatman® Elutrap® Electroelution Systems (GE Healthcare), or other devices may be used to electroelute the genetic material directly from the solid medium into solution.
  • the genetic material may be directly electroeluted from the solid medium onto a capture surface.
  • the capture surface may be on a blotting membrane (e.g., diethylaminoethyl cellulose (DEAE)).
  • DEAE diethylaminoethyl cellulose
  • the extracted genetic material may be analyzed (block 28).
  • the genetic material extracted from the sample-containing solid medium includes high molecular weight DNA optimally of at least 10 kilobases with few nicks present. Indeed, the DNA extracted in accordance with the present approaches may approach 40 kilobases.
  • DNA extraction occurs in the absence of detergents. If detergent were used in extracting the DNA from the solid medium, additional steps would typically be performed to remove the detergent before any subsequent analysis of the extracted DNA could occur.
  • the extraction of DNA at high temperatures e.g., 95° C.
  • the extracted DNA may be used in a variety of analyses including polymerase chain reaction (PCR), single-nucleotide polymorphism analysis, real-time PCR, and other downstream uses of the DNA.
  • FIGS. 2 and 3 illustrate examples of the electroelution of DNA directly from the sample-containing solid medium into electrophoresis gels.
  • FIG. 2 depicts a SYBR® Gold (Invitrogen) stained electrophoresis gel of DNA electroeluted from solid media using pulsed-field gel electrophoresis. Electrophoresis was in a 1% agarose gel, 0.5 ⁇ TE, ph 8.0 for 18 hours at 6 V/cm with a switch angle of 120° and switch time from 50-90 seconds. 20 ⁇ L of Jurkat cells (suspended at 2 ⁇ 10 6 cells/mL) were applied to either Whatman® FTA® or FTA® Elute paper and dried as described above.
  • SYBR® Gold Invitrogen
  • Lane 1 Saccharomyces cerevisiae chromosomal DNA size marker (BioRad); Lane 3: FTA® paper soaked for 5 minutes in TE, ph 8.0; Lane 5: FTA® paper soaked for 5 minutes in FTA® purification reagent (two times) and soaked for 5 minutes in TE buffer, ph 8.0 (two times); Lane 7: FTA® Elute paper soaked in water for 5 minutes; Lane 9: FTA® Elute paper soaked in water for 5 minutes, then vortexed for 15 seconds; Lane 11: FTA® Elute paper soaked in water for 5 minutes, then eluted into 95° C.
  • Lane 12 FTA® Elute paper soaked in water for 5 minutes, then eluted into 95° C. water for 25 minutes, and vortexed for 60 seconds; and Lane 14: Saccharomyces cerevisiae chromosomal DNA size marker (BioRad). After pulsed-field gel electrophoresis, the DNA was stained with SYBR® Gold and the gel was imaged on a TyphoonTM Imager fluorescent scanner.
  • FIG. 2 demonstrate the electroelution of high molecular weight DNA from the samples of Jurkat cells directly from the solid medium (i.e., FTA® or FTA® Elute paper) into the electrophoresis gel.
  • the electroeluted DNA is less than 225 kilobases but estimated to be approximately 40 kilobases.
  • FIG. 2 also demonstrates the absence of high molecular weight DNA when attempting to elute the DNA under high temperature conditions (e.g., 95° C.).
  • a buffer solution e.g., TE buffer or FTA® purification reagent
  • FIG. 3 illustrates similar results using alkaline gel electrophoresis.
  • FIG. 3 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using alkaline standard gel electrophoresis. Electrophoresis was in a 1% agarose gel, 30 mM NaOH, 1 mM EDTA, for 2 hours at 150 mA. During electrophoresis the gel was covered in a glass plate to prevent diffusion out of the gel. As above, 20 ⁇ L of Jurkat cells (suspended at 2 ⁇ 10 6 cells/mL) were applied to Whatman® FTA® paper and dried as described above.
  • the results in FIG. 3 also demonstrate the electroelution of high molecular weight DNA from the samples of Jurkat cells directly from the solid medium (i.e., FTA® or FTA® Elute paper) into the electrophoresis gel.
  • the electroeluted DNA is greater than 10 kilobases.
  • a buffer solution e.g., TE buffer or FTA® purification reagent
  • water is needed in order to electroelute high molecular weight DNA from the solid medium directly into the electrophoresis gel.
  • a buffer solution e.g., TE buffer or FTA® purification reagent
  • the fact that the large DNA is also present on an alkaline gel indicates that the electroeluted DNA contains few nicks. As the DNA in an alkaline gel is single stranded, any nicks in the backbone would result in DNA fragments.
  • FIG. 4 is a flow chart illustrating a method 30 for extracting genetic material (e.g., DNA) from a fixed tissue sample stored on a solid medium.
  • the fixed samples may also include fixed cells (e.g., derived from tissues or cell culture lines).
  • tissue is collected from a source (e.g., a mouse or human) (block 32).
  • a fixing agent such as formalin, paraformaldehyde, or other fixing agent, according to methods know to the art.
  • the genetic material e.g., DNA
  • the fixed tissue samples may be dehydrated (block 36) in a series of ethanol washes with the ethanol sequentially increasing in concentration. The dehydrated samples may then be washed in xylene and embedded in paraffin.
  • the sample is rehydrated (block 38) in a series of ethanol washes sequentially decreasing in concentration.
  • the fixed tissue sample is rehydrated sequentially for 5 minutes each in 100% ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and distilled water (or T.E.).
  • the rehydrated sample may be incubated overnight in 1 M potassium thiocyanate.
  • the fixed tissue sample, rehydrated or not, may be lyzed (block 40), e.g., with a protease, prior to application to the solid medium.
  • the fixed tissue sample may be lyzed in a digest buffer solution including Proteinase K (0.5 mg/mL), 50 mM Tris at pH 7.4, 10 mM EDTA, 0.5% SDS, and 50 mM NaCl at 55° C. for at least three hours.
  • the fixed tissue sample may be lysed via chemicals present on the solid medium as described above.
  • the sample of fixed is applied to the solid medium via such methods as rubbing the sample against the media, pressing the sample against the media, etc.
  • the solid medium is as described above.
  • the solid medium includes chemicals to preserve the genetic material on the solid medium.
  • the solid medium includes chemicals that lyze the fixed tissue sample as described above. Any liquid within the applied tissue sample evaporates after application. Drying of the sample on the solid medium (block 44) occurs after application.
  • the solid medium containing the sample may dry overnight in a desiccator.
  • the solid medium containing the sample may be encased in a protective material (e.g., a plastic film) to further preserve the genetic material.
  • a portion of the solid medium containing the sample is removed for electroeluting the genetic material (e.g., DNA) from this portion.
  • the entire sample may be used.
  • the portion of the solid medium containing the sample may be rinsed to remove the chemicals (block 46).
  • rinsing the portion of the sample-containing solid medium includes soaking the solid medium portion in a buffer solution.
  • the buffer solution may include an alkaline buffer solution (e.g., TE) or FTA® purification reagent.
  • the sample-containing solid medium portion may be soaked for a fixed time (e.g., 5 minutes each soaking) twice in the FTA® purification reagent and for a fixed time (e.g., 5 minutes each soaking) twice in the alkaline buffer solution.
  • the rinses may occur only in the alkaline buffer solution, only in the FTA® purification reagent, or only in water as described above.
  • electroelution e.g., electroelution in dialysis tubing
  • rinsing of the sample-containing solid medium may not be necessary.
  • the solid medium portion with the fixed sample may be treated with enzymes (block 48) to reduce or eliminate non-DNA contaminants (e.g., proteins, lipids, carbohydrates, etc.).
  • enzymes e.g., proteins, lipids, carbohydrates, etc.
  • an enzymatic solution may include hydrolytic enzymes such as proteases, lipases, and/or glycoside hydrolases.
  • the cross-linking between the genetic material and protein may be reversed (block 50) subsequent to applying the fixed tissue sample to the solid medium.
  • a crosslink repair buffer including a primary amine (e.g., bicine, pH 8.5) or other nucleophile with a pH higher than 7.0 (preferably with a pH between 7.5-10).
  • the sample-containing solid medium portion is incubated in the crosslink repair buffer (e.g. at 65° C. for 20 hours).
  • the primary amine or nucleophile reacts with the portion of the crosslink that comes from the aldehyde that originally formed the cross-links (between the genetic material and/or the protein) and reverses these cross-links.
  • a sulfhydryl reagent can be used in place of amine, which will also react with the portion of the crosslink that is derived from the aldehyde. This reaction occurs more rapidly than amine-based nucleophile when performed at a pH below 7.
  • the reversal of the cross-linking may occur prior to application of the fixed tissue sample to the solid medium. Further, prior to electroeluting genetic material from the sample-containing solid medium portion, the genetic material (e.g., DNA) may be repaired while fixed in position on the solid medium (block 52) as described above.
  • the genetic material is directly electroeluted from the solid medium to a subsequent medium (block 54) as described above to obtain high molecular weight DNA of at least 10 kilobases.
  • the extracted genetic material may then be analyzed (block 56) in a variety of ways (e.g., PCR) as demonstrated in FIG. 5 .
  • FIG. 5 illustrates the quality of the DNA extracted from formalin-fixed tissue samples on solid media following crosslink reversal and DNA repair as described above.
  • FIG. 5 depicts a SYBR® Gold stained electrophoresis gel, using a vertical polyacrylamide gel electrophoresis (PAGE) system, of multiplex PCR amplification of DNA that has been extracted from a human prostate sample fixed in formalin and applied to Whatman® FTA® paper as described above. Electrophoresis was in a 6% TBE (Tris-Borate-EDTA)-UREA gel and 1 ⁇ TBE for 40 minutes at 160V. The fixed human prostate sample was processed as described above (e.g., rehydrated or lysed) and applied to the FTA® paper.
  • TBE Tris-Borate-EDTA
  • the samples on the FTA® paper were then subjected to crosslink reversal and/or DNA repair (with or without endonuclease IV (Endo IV)).
  • the solid media is added directly to a PCR reaction containing 3 sets of PCR primers.
  • the sequences of the primers are as follows: 5′ CTCACCCTGAAGTTCTCAGG 3 ′ (primer 1), 5′ CCTCAAGGGCACCTTTGCCA 3′ (primer 2), 5′ GTCTACCCTTGGACCCAG 3′ (primer 3), and 5′ GATGAAGTTGGTGGTGAGG 3′ (primer 4).
  • the PCR primers are designed to produce products of 78 base pairs (primers 1 and 2), 218 base pairs (primers 1 and 3), and 380 base pairs (primers 1 and 4) if high molecular weight human DNA is present.
  • Amplification conditions were as follows: step 1: 95° C. for 7 minutes; step 2 (for 15 cycles): 95° C. for 1 minute, 69° C. for 1 minute, and 72° C. for 1 minute; step 3 (for 15 cycles): 95° C. for 1 minute, 63° C. for 1 minute, and 72° C. for 1 minute; step 4: 72° C. for 10 minutes; and then step 5: 4° C.
  • the samples shown in FIG. 5 are as follows:
  • Lane 1 low molecular weight DNA ladder (New England Biolabs); Lane 3: purified human DNA (Male, Applied Biosystems); Lane 4: no DNA control; Lane 5: purified human DNA treated with repair reaction (without Endo IV); Lane 6: purified human DNA treated with repair reaction (with Endo IV); Lane 8: formalin-fixed human prostate sample on FTA® paper with crosslink reversal and repair reactions (without Endo IV); Lane 9: formalin-fixed human prostate sample on FTA® paper with repair reaction only (with Endo IV); Lane 10: formalin-fixed human prostate sample on FTA® paper with crosslink reversal and repair reactions (with Endo IV); Lane 11: formalin-fixed human prostate sample on FTA® paper with repair reaction only (with Endo IV); Lane 12: formalin-fixed human prostate sample on FTA® paper with crosslink reversal reaction only; Lane 13: formalin-fixed human prostate sample on FTA® paper without crosslink reversal and repair reactions; and Lane 15: low molecular weight DNA ladder. After PAGE gel
  • FIG. 5 demonstrate the appearance of the higher molecular weight PCR products (218 base pairs and 380 base pairs) when the sample on the FTA® paper has been treated to reverse cross-links and to repair DNA (Lane 10) as described above. All other combinations of treatment fail to produce these higher molecular weight PCR products. While the DNA in this experiment has not been eluted from the FTA® paper prior to PCR, it can be appreciated that if the repaired sample still located on the FTA® paper can be used for PCR amplification of higher molecular weight targets, this result in combination with the ability to elute DNA from FTA® paper would allow for a greater range of DNA interrogation experiments to be performed on such samples.
  • FIGS. 6 and 7 illustrate the benefit of rehydrating fixed samples prior to electroelution from the FTA paper.
  • FIGS. 6 and 7 depict a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using native gel electrophoresis. Electrophoresis was in a 0.8% agarose TBE gel for 80 minutes at 110 volts. The samples shown in FIG.
  • Lane 1 1 kb DNA ladder (New England Biolabs); Lane 2: 0.5 mm slice of formalin-fixed human prostate sample applied directly to FTA® paper, dried, and rinsed in water and electroeluted; Lane 3: 20 ⁇ L of Jurkat cells (suspended at 2 ⁇ 10 6 cells/mL) were applied to FTA® paper, dried, and electroeluted; and Lane 4: purified human DNA (Male, Applied Biosystems). The electroeluted samples shown in FIG.
  • Lane 7 are as follows: Lane 1: purified human DNA (Male, Applied Biosystems); Lane 2: 20 ⁇ L of Jurkat cells (suspended at 2 ⁇ 10 6 cells/mL) were applied to FTA® paper, and dried; Lane 3: an approximately 0.5 mm slice of formalin-fixed human prostate sample was rehydrated as described above and applied to FTA® paper, dried, and rinsed in water; and Lane 4: approximately 0.5 mm of formalin-fixed human prostate sample was scraped off the paraffin block as a series of flakes, then rehydrated as described above, applied to FTA® paper, dried, and rinsed in water. After TBE-gel electrophoresis, the DNA was stained with SYBR® Gold and the gel was imaged on a TyphoonTM Imager fluorescent scanner.
  • FIGS. 6 and 7 in particular a comparison between lane 2 of FIG. 6 and lanes 3 and 4 of FIG. 7 , clearly indicate that the rehydration process aids in the retrieval of DNA from fixed samples.
  • the inclusion of steps to rehydrate tissue samples that have been dehydrated in preparation for fixation and for paraffin embedding increases the yield of genetic material from the fixed sample upon electroelution.
  • FIG. 8 illustrates the effect of DNA repair on the molecular weight of DNA obtained from fixed samples that have been electroeluted from FTA paper.
  • FIG. 8 depicts a SYBR® Gold stained electrophoresis gel of DNA electroeluted from solid media using native gel electrophoresis. Electrophoresis was in a 0.8% agarose TBE gel for 80 minutes at 110 volts. Samples from formalin-fixed human lung (ILS 19279-A04) were applied to FTA® paper as described above. Cross-links were repaired in 100 mM bicine pH 9.5 and/or treated with a DNA repair reaction in certain samples as described above. The samples were stopped at different stages of the reactions. The samples shown in FIG. 8 are as follows:
  • Lane 1 1 kb DNA ladder (New England Biolabs); Lane 2: post rehydration (i.e., ethanol and T.E. washes); Lane 3: post FTA® purification reagent washes; Lane 4: post Proteinase K digestion; Lane 5: post crosslink reversal reaction conducted at 65° C.; Lane 6: post crosslink reversal reaction conducted at room temperature; Lane 7: post DNA precipitation (plus crosslink reversal reaction conducted at 65° C.); Lane 8: post DNA precipitation (plus crosslink reversal reaction conducted at room temperature); Lane 9: post DNA repair reaction (plus crosslink reversal reaction conducted at 65° C.); and Lane 10: post DNA repair reaction (plus crosslink reversal reaction conducted at room temperature).
  • FIG. 8 demonstrate the appearance of high molecular weight material in lanes 9 and 10 (indicated by the arrow) only after the DNA repair reaction has been used to repair the DNA that is still attached to the FTA® paper.
  • the DNA is repaired while it is still attached to the FTA® paper, before the DNA has been subjected to the forces involved in elution of the DNA from the FTA® paper and the forces that are applied to DNA in solution.
  • This prevents strands of DNA which contain nicks that are located relatively near each other, but on opposite sides, from separating. By repairing these highly nicked and gapped strands, they are prevented from becoming double stranded breaks, and the resulting product is higher molecular weight DNA.
  • the increase in molecular weight provides evidence that the DNA repair reaction was able to repair nicks and gaps in the DNA.
  • inventions include extracting genetic material (e.g., high molecular weight DNA of at least 10 kilobases) directly from the solid medium via electroelution.
  • the extraction of genetic material occurs in the absence of detergent, thus eliminating additional steps necessary for the removal of detergent prior to any subsequent analysis of the genetic material.
  • the extraction of genetic material avoids the use of high temperatures (e.g., 95° C.), thus allowing the extraction of minimally fragmented DNA.
  • the extraction of genetic material, via electroelution directly from the solid medium applies to both fixed and non-fixed samples.
  • the genetic material may be processed while still on the solid medium. For example, genetic material may be repaired and/or cross-links reversed in fixed cells between the genetic material and protein.
  • the solid medium provides a single platform for the extraction of genetic material and related processes (e.g., DNA repair) minimizing the work for the user.

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US9040675B2 (en) 2012-04-30 2015-05-26 General Electric Company Formulations for nucleic acid stabilization on solid substrates
US9040679B2 (en) 2012-04-30 2015-05-26 General Electric Company Methods and compositions for extraction and storage of nucleic acids
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US10625242B2 (en) 2012-04-30 2020-04-21 General Electric Company Substrates and methods for collection, stabilization and elution of biomolecules
US9333463B2 (en) 2013-07-26 2016-05-10 General Electric Company Devices and systems for elution of biomolecules
US9999856B2 (en) 2013-07-26 2018-06-19 General Electric Company Methods for electroelution of biomolecules
US11266337B2 (en) 2015-09-09 2022-03-08 Drawbridge Health, Inc. Systems, methods, and devices for sample collection, stabilization and preservation
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