US008927214B2
`
`US8,927,214 B2
`(10) Patent No.:
`az) United States Patent
`Yarnall et al.
`(45) Date of Patent:
`Jan. 6, 2015
`
`
`(54) METHODS AND COMPOSITIONS FOR DUAL
`EXTRACTION OF PROTEIN AND NUCLEIC
`ACID
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`(75)
`
`Inventors: Michele Susan Yarnall, Durham, NC
`(US); Wenling Wang, Durham, NC
`(US); Karen Moore, Durham, NC (US);
`Mary Fielder, Durham, NC (US)
`
`(73) Assignee: Syngenta Participations AG, Basel
`(CH)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`US.C. 154(b) by 152 days.
`
`(21) Appl. No.: 13/450,039
`
`(22)
`
`Filed:
`
`Apr. 18, 2012
`
`(65)
`
`Prior Publication Data
`
`US 2013/0280718 Al
`
`Oct. 24, 2013
`
`(51)
`
`Int. Cl.
`C12Q 1/68
`C1I2P 19/34
`(52) U.S.CL.
`USPC iececeesscctesereescersenesenecneanees 435/6.1; 435/91.2
`
`(2006.01)
`(2006.01)
`
`(58) Field of Classification Search
`USPC viececseeseccrescnseecensesssenscnsensceesesesanecnees 435/91.2
`See application file for complete search history.
`
`3/2001 Lader
`6,204,375 Bl
`7/2003 Andersonet al.
`2003/0131377 Al
`4/2011 Ehwaldet al.
`2011/0098462 Al
`10/2011 Hollanderetal.
`2011/0244468 Al
`OTHER PUBLICATIONS
`
`Protein Buffers, Luminex Corporation website (2007) online,
`Retrieved on Jun. 8, 2012 from the Internet (2 pages).
`Invitrogen: Guanidine Isothiocyanate Solution (2006) online,
`Retrieved on Jun. 8, 2012 from the Internet (4 pages).
`Tolosa et al. “Column-Based Method to Simultaneously Extract
`DNA, RNA,andProteins from the Same Sample” Bio Techniques
`43:799-804 (Dec. 2007).
`International Search Report and Written Opinion for International
`Application No. PCT/US12/34086, dated Jul. 13, 2012 (8 pages).
`
`Primary Examiner — Cynthia B Wilder
`(74) Attorney, Agent, or Firm — Myers Bigel Sibley &
`Sajovec, PA
`
`ABSTRACT
`(57)
`The present invention provides a methodofisolating nucleic
`acid and protein from the same biological sample, compris-
`ing,
`in the following order: a) disrupting the biological
`sample; b) contacting the disrupted biological sample of (a)
`with a protein lysis buffer that lacks any component that
`denatures or reduces protein to produce a first lysate; c) cen-
`trifuging thefirst lysate of (b) to producea first supernatant
`containing protein and a pellet containing nucleic acid; d)
`removing the first supernatantof(c), thereby isolating protein
`from the biological sample; e) contacting the pellet of (d) with
`nucleic acid lysis buffer to produce a secondlysate; f) centri-
`fuging the secondlysate of (e) to produce a second superna-
`tant containing nucleic acid; and g) removing the second
`supermatant of (f), thereby isolating nucleic acid from the
`same biological sample.
`11 Claims, No Drawings
`
`Inari Exhibit 1073
`Inari Exhibit 1073
`Inari v. Pioneer
`Inari v. Pioneer
`
`

`

`US 8,927,214 B2
`
`1
`METHODS AND COMPOSITIONS FOR DUAL
`EXTRACTION OF PROTEIN AND NUCLEIC
`ACID
`
`FIELD OF THE INVENTION
`
`The invention relates to methods and compositions for
`extraction of protein and nucleic acid from a single tissue
`sample.
`
`BACKGROUNDOF THE INVENTION
`
`Biological samples, e.g., plant tissue samples, are routinely
`tested for the presence of the gene (DNA) and whether the
`gene product (protein) is expressed. Currently, tissues from
`organisms, such as the plants or animals, have to be sampled
`separately for each ofthese tests. If both the protein and DNA
`can be extracted from the same sample, then a significant
`savings would be achieved in both consumable costs as well
`as time and labor savings. Methodsexist for simultaneous
`extraction of DNA/RNA/protein but the resulting protein,
`extracted with buffers that denature the protein, is not suitable
`for measurement in quantitative methods such as enzyme
`linked immunosorbent assay (ELISA); it can only be mea-
`sured by methods that can employ denatured proteins(e.g.,
`western blot). Existing methods isolate the nucleic acid com-
`ponentsfirst to prevent any potential degradation ofthis mate-
`rial, but this process will cause the denaturation of the pro-
`teins in the mixture. Thus, the present invention overcomes
`previous shortcoming in the art by providing compositions
`and methods for dual extraction of undenatured protein and
`nucleic acid from a single tissue sample.
`
`SUMMARYOF THE INVENTION
`
`invention provides a method of isolating
`The present
`nucleic acid and protein from the same biological sample,
`comprising, in the following order: a) disrupting the biologi-
`cal sample; b) contacting the disrupted biological sample of
`(a) with a protein lysis buffer that lacks any component that
`denatures or reduces protein to producea first lysate; c) cen-
`trifuging the first lysate of (b) to producea first supernatant
`containing protein and a pellet containing nucleic acid; d)
`removingthe first supernatantof (c), thereby isolating protein
`from the biological sample; e) contacting the pellet of (d) with
`nucleic acid lysis buffer to produce a secondlysate; f) centri-
`fuging the secondlysate of (e) to produce a second superna-
`tant containing nucleic acid; and g) removing the second
`supernatant of (f), thereby isolating nucleic acid from the
`same biological sample.
`These and other aspects of the invention are set forth in
`moredetail in the description of the invention below.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`This description is not intended to be a detailed catalog of
`all the different ways in which the invention may be imple-
`mented, or all the features that may be addedto the instant
`invention. For example, features illustrated with respect to
`one embodiment may be incorporated into other embodi-
`ments, and features illustrated with respect to a particular
`embodiment maybe deleted from that embodiment. In addi-
`tion, numerous variations and additions to the various
`embodiments suggested herein will be apparent to those
`skilledin the art in light ofthe instantdisclosure, which do not
`depart from the instant
`invention. Hence,
`the following
`descriptions are intended to illustrate some particular
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`embodiments ofthe invention, and notto exhaustively specify
`all permutations, combinations and variations thereof.
`Unless otherwise defined, all technical and scientific terms
`used herein have the same meaning as commonly understood
`by one of ordinary skill in the art to which this invention
`belongs. The terminology used in the description ofthe inven-
`tion herein is for the purpose of describing particular embodi-
`ments only andis not intendedto be limiting ofthe invention.
`All publications, patent applications, patents, and other ref-
`erences mentioned herein are incorporated by reference in
`their entirety.
`The present invention is based on the unexpected discovery
`that both protein (e.g., undenatured protein) and nucleic acid
`of good quality for analysis can be extracted from the same
`biological sample. Theprotein thatis isolatedis not denatured
`and can be tested for functional activity or measured in other
`assays without dilution to remove the denaturation agents.
`The nucleic acid that is isolated retains its integrity through
`the protein isolation process, during which nucleases are
`present. Thus in one embodiment, the present invention pro-
`vides a method ofisolating nucleic acid and protein (e.g.,
`undenatured protein or protein in its native configuration)
`from the samebiological sample, comprising, in the follow-
`ing order: a) disrupting the biological sample; b) contacting
`the disrupted biological sample of (a) with a protein lysis
`buffer that lacks any component that denatures or reduces
`protein to producea first lysate; c) centrifuging thefirst lysate
`of (b) to producea first supernatant containing protein anda
`pellet containing nucleic acid; d) removing thefirst superna-
`tant of (c), thereby isolating protein from the biological
`sample; e) contacting the pellet of (d) with nucleic acid lysis
`buffer to produce a secondlysate; f) centrifuging the second
`lysate of (e) to produce a second supernatant containing
`nucleic acid; and g) removing the second supernatant of (1,
`thereby isolating nucleic acid from the same biological
`sample.
`In some embodiments of the methods described herein,
`nonlimiting examples of waysto disrupt a biological sample
`include physical disruption (e.g., mortar andpestle, shearing,
`sonication, processors, homogenization by high pressure,fil-
`tration), freeze/thaw cycles to allow for cell lysis, permeabi-
`lization, etc., as are knownin theart.
`In some embodiments of the method above, the protein
`lysis buffer can be Protein Lysis Buffer A (phosphate buffered
`saline with 0.05% Tween-20) or BB-PVP-Tw buffer (borate
`buffer 0.1 M, pH 7.5, 0.2% polyvinylpyrrolidone (PVP),
`0.5% Tween-20). The lysis buffer employed in the methods of
`this invention maintains the biological structure and function
`of the protein in the biological sample and does not denature
`or reduce the protein. Other biological buffers that can be
`used in the methods of this invention include buffers that
`maintain the pH ofthe solution in a range from about4.5 to
`about 9.0 (e.g., Tris, MES, HEPES, MOPS, PIPES, Imida-
`zole, Citrate and others as are know in theart).
`In particular embodiments, the protein lysis buffer lacks
`any (e.g., any detectable amount) componentthat denatures
`or reduces protein. Nonlimiting examples of such compo-
`nents include sodium dodecyl sulfate (SDS), urea, guanidine,
`a buffer with a pH above about 9.0 or pH below about 4.5,
`2-mercaptoethanol anddithiothreitol.
`In some embodiments of the method above, the nucleic
`acid lysis buffer is PA lysis buffer (4 M guanidinethiocyanate,
`10mM Tris, pH 7.5), LG extraction buffer (200 mM Tris HCl,
`pH 8.5, EDTA 25 mM, 1% SDS), Promega Lysis Buffer A, or
`CTABbuffer (2% CTAB, 100 mM Tris HCl, pH 8.0, 20 mM
`EDTA,1.4 M NaCl, 40 mM 2-mercaptoethanol), NaOH and
`SDSor Tween.
`
`

`

`US 8,927,214 B2
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`0
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`In various embodiments, the isolation method above can
`further comprise the step of detecting and/or quantitating
`protein in the supernatant(e.g., the first supernatant) by any
`method known for detecting and/or quantitating protein.
`Nonlimiting examples of such methods include ELISA,
`BCA, Bradford assay, western blot, immunoprecipitation,
`immunoassay, chromatographic methods such as HPLC,
`enzymatic methods, functional assays and any other method
`knownto detect/quantitate protein.
`In some embodiments, the method described above can
`further comprise the step of analyzing(e.g., detecting and/or
`quantitating) the nucleic acid (e.g., the nucleic acid in the
`second supernatant) by any methodavailable to detect and/or
`quantitate nucleic acid (e.g., DNA, RNAor both) in a sample.
`Nonlimiting examples of such methodsinclude electrophore-
`sis, amplification reactions such as the polymerase chain
`reaction (PCR), realttme PCR, TaqMan assays, etc., as are
`knownin the art.
`In the methods of this invention, the extracts of this inven-
`tion can be prepared as described herein and can bestoredat
`about 4° C. (e.g., about 2° C., 3° C., 4° C., 5° C., 6° C., ete.)
`with or without the PBST removedfor upto about 24 hrs(e.g.,
`12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
`29, 30, 31, 32, 33, 34, 35, 36 hrs, etc.) without loss (e.g., a
`detectable loss or measurable loss of less than about 50%,
`40%, 30%, 20%, 10%, 5%, 2%, 1% or 0% according to the
`assays described herein) of protein and/or without degrada-
`tion (e.g., a detectable or measurable degradation of less than
`about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or 0% accord-
`ing to the assays described herein) of DNA.
`In some embodiments of this invention, the biological
`sample can be from a plant. Nonlimiting examples ofbiologi-
`cal sample sources from plant includeleaf tissue, seed tissue,
`root tissue, stem tissue, flowertissue, etc.
`The methods of this invention can also be employed to
`isolate protein (e.g., non-denatured and non-reducedprotein)
`and nucleic acid from a biological sample from an animal.
`Sources of animal biological samples, such as blood and
`blood components, body fluids, tissues, etc., are well known
`in the art.
`
`The plant of this invention can be any plant from which a
`tissue sample can be obtained for use in the methods
`described herein. Nonlimiting examples include field crops
`such as maize, soy, sugarcane, sugarbeet, cotton,rice, peanut,
`sunflowerand tobacco; pasture and hay crops such asalfalfa,
`pasture grasses andsilage; and fruit and vine crops, such as
`apples, apricots, cherries, peaches, pears, plums, citrus,
`grapes, pecans, walnuts, avocados, bananas and kiwis.
`In particular embodiments of this invention, the method
`described herein (having steps (a) through (g)) can further
`comprise the steps of: h) contacting the supernatant of (g)
`with magnetic beads in wells of a multiwellplate; 1) position-
`ing the multiwell plate on a magnet for a period of time
`sufficient for nucleic acid present in the supernatant to bind
`the magnetic beads in the wells of the multiwell plate; j)
`removing the supernatant from the wells of the multiwell
`plate; k) washing the magnetic beads with wash buffer[e.g.,
`50% PA wash buffer (125 mM Tris pH 7.5, 25 mM EDTA,
`500 mM NaC)), 25% 2-isopropanol and 25% ethanol (95%)];
`1) positioning the multiwell plate on the magnet; m) removing
`the wash buffer; n) repeating steps (k) and (1) at least two
`times; 0) drying the magnetic beads under conditions
`wherebythe ethanol in the wash buffer evaporates; p) adding
`elution buffer to the wells of the multiwell plate to elute the
`nucleic acid from the magnetic beads; q) positioning the
`multiwell plate on the magnet for a period of time sufficient
`for the magnetic beads to collect at the bottom of the wells of
`
`4
`the multiwell plate; r) optionally removingthe elution buffer
`containing the nucleic acid from the wells of the multiwell
`plate; and s) contacting the elution buffer containing the
`nucleic acid with reagents for analysis (e.g., detection and/or
`quantitation) of the nucleic acid.
`In some embodiments, step (r) can be carried out and the
`elution buffer can be removed from the wells ofthe multiwell
`
`plate containing the magbeads andtransferred to another
`plate or container or vessel for analysis of the nucleic acid in
`the elution buffer according to methods well knownintheart.
`Alternatively, the elution buffer can remain in the wells ofthe
`multiwall plate containing the magbeads for analysis of the
`nucleic acid in the elution buffer according to methods well
`knownin the art.
`In some embodiments of the methods described above, the
`elution buffer can be water, Promegaelution buffer, low TE
`buffer (1 mM Tris, 0.1 mM EDTA,pH 8.0), or TE buffer (10
`mM Tris-Cl, 1 mM EDTA,pH 8.0).
`In various embodiments, the reagents for analyzing the
`nucleic acid are reagents for amplification of nucleic acid, as
`described herein.
`
`66
`
`
`DEFINITIONS
`P66,
`“an” or “the” can mean one or more
`“a.”
`As used herein,
`than one. For example, a cell can mean a single cell or a
`multiplicity of cells.
`As used herein, “and/or” refers to and encompasses any
`and all possible combinations ofone or more ofthe associated
`listed items, as well as the lack of combinations when inter-
`preted in the alternative (or).
`Further, the term “about,” as used herein whenreferring to
`a measurable value such as an amount of a compoundor
`agent, dose, time, temperature, and the like,
`is meant to
`encompass variations of +20%, +10%, +5%, +1%, +0.5%, or
`even +0.1% of the specified amount.
`As used herein, the transitional phrase “consisting essen-
`tially of’ meansthat the scope of a claim isto be interpreted
`to encompass the specified materials or steps recited in the
`claim and those that do not materially affect the basic and
`novel characteristic(s) of the claimed invention. Thus, the
`term “consisting essentially of when used in a claim ofthis
`invention is not intendedto be interpreted to be equivalent to
`“comprising.”
`The term “plant” is intended to encompass plants at any
`stage of maturity or development, as well as any tissues or
`organs (plant parts) taken or derived from any such plant
`unless otherwise clearly indicated by context. The present
`invention also includes transgenic seeds produced by the
`transgenic plants of the present invention. In one embodi-
`ment, the seeds are true breeding for an increased resistance
`to nematode infection as compared to a wild-type variety of
`the plant seed. In particular embodimentsofthe invention, the
`plant is a soybean plant.
`As used herein, the term “plant part” includes but is not
`limited to pollen, seeds, branches, fruit, kernels, ears, cobs,
`husks, stalks, root tips, anthers, stems, roots, flowers, ovules,
`stamens, leaves, embryos, meristematicregions, callus tissue,
`anther
`cultures,
`gametophytes,
`sporophytes,
`pollen,
`microspores, protoplasts, hairy root cultures, and the like.
`plantcells including plant cells that are intact in plants and/or
`parts of plants, plant protoplasts, plant tissues, plant cell
`tissue cultures, plant calli, plant clumps, andthe like. Further,
`as used herein, “plant cell” refers to a structural and physi-
`ological unit ofthe plant, which comprises a cell wall and also
`mayrefer to a protoplast. Thus, as used herein,a “plant cell”
`includes, butis not limitedto, a protoplast, gamete producing
`
`

`

`US 8,927,214 B2
`
`5
`cell, and a cell that regenerates into a whole plant. Tissue
`culture of varioustissues of plants and regeneration of plants
`therefrom is well knownintheart.
`
`A plantcell ofthe present invention can be in the form of an
`isolated single cell or can be a cultured cell or can be a part of
`a higher-organized unit such as, for example,a plant tissue or
`a plant organ.
`Also as used herein, the terms “‘nucleic acid,” “nucleic acid
`molecule, “nucleotide sequence”and “polynucleotide”refer
`to RNA or DNAthat is linear or branched, single or double
`stranded, or a hybrid thereof. The term also encompasses
`RNA/DNAhybrids. When dsRNAis produced synthetically,
`less commonbases, such as inosine, 5-methylcytosine, 6-me-
`thyladenine, hypoxanthine and others can also be used for
`antisense, dsRNA,and ribozymepairing. For example, poly-
`nucleotides that contain C-5 propyne analogues of uridine
`and cytidine have been shownto bind RNAwith highaffinity
`and to be potent antisense inhibitors of gene expression.
`Other modifications, such as modification to the phosphodi-
`ester backbone, orthe 2'-hydroxyin the ribose sugar group of
`the RNA can also be made.
`
`Asused herein, the term “nucleotide sequence”refers to a
`heteropolymerof nucleotides or the sequenceof these nucle-
`otides from the 5' to 3' end of a nucleic acid molecule and
`
`includes DNA or RNA molecules, including cDNA, a DNA
`fragment, genomic DNA,synthetic (e.g., chemically synthe-
`sized) DNA, plasmid DNA, mRNA,andanti-sense RNA, any
`ofwhich can be single stranded or double stranded. The terms
`“nucleotide sequence” “nucleic acid’ “nucleic acid mol-
`ecule,” “oligonucleotide” and “polynucleotide”are also used
`interchangeably herein to refer to a heteropolymerof nucle-
`otides. Nucleic acid sequences provided herein are presented
`herein in the 5' to 3' direction, from left to right and are
`represented using the standard code for representing the
`nucleotide characters as set forth in the U.S. sequencerules,
`37 CFR §§1.821-1.825 and the World Intellectual Property
`Organization (WIPO) Standard ST.25.
`A “nucleic acid”or “nucleic acid molecule”is a nucleotide
`
`sequence (either DNA or RNA) that is present in a form or
`setting that is different from that in whichit is foundin nature
`andis not immediately contiguous with nucleotide sequences
`with which it is immediately contiguous (one on the 5' end
`and one on the 3' end) in the naturally occurring genomeofthe
`organism from which it
`is derived. Accordingly,
`in one
`embodiment, an isolated nucleic acid includes someorall of
`the 5' non-coding (e.g., promoter) sequences that are imme-
`diately contiguous to a coding sequence. The term therefore
`includes, for example, a recombinant nucleic acid that is
`incorporated into a vector, into an autonomously replicating
`plasmidor virus, or into the genomic DNA ofa prokaryote or
`eukaryote, or which exists as a separate molecule (e.g., a
`cDNA or a genomic DNA fragment produced by PCR or
`restriction endonuclease treatment), independent of other
`sequences. Thus, a nucleic acid molecule foundin nature that
`is removedfrom its native environmentand transformed into
`a plantis still considered “isolated” even when incorporated
`into a genome of the resulting transgenic plant.
`It also
`includes a recombinant nucleic acid that is part of a hybrid
`nucleic acid encoding an additional polypeptide or peptide
`sequence.
`The term “isolated” can further refer to a nucleic acid,
`nucleotide sequence, polypeptide, peptide or fragmentthat is
`substantially free of cellular material, viral material, and/or
`culture medium (e.g., when produced by recombinant DNA
`techniques), or chemical precursors or other chemicals (e.g.,
`when chemically synthesized). Moreover, an “isolated frag-
`ment”is a fragmentof a nucleic acid, nucleotide sequence or
`
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`polypeptidethat is not naturally occurring as a fragment and
`would not be found as such in the natural state. “Isolated”
`
`does not meanthat the preparation is technically pure (homo-
`geneous), but it is sufficiently pure to provide the polypeptide
`or nucleic acid in a form in which it can be used for the
`
`intended purpose.
`In representative embodiments of the invention, an “iso-
`lated” nucleic acid, nucleotide sequence, and/or polypeptide
`is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,
`60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure
`(w/w) or more. In other embodiments, an “isolated” nucleic
`acid, nucleotide sequence, and/or polypeptide indicates that
`at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold,
`10,000-fold, 100,000-fold or more enrichmentof the nucleic
`acid (w/w) is achieved as compared withthe starting material.
`The present
`invention also provides a kit comprising
`reagents, buffers, diluents, devices and/or instruments, etc., to
`carry out the methods of this invention. It would be well
`understood by one of ordinary skill in the art that the kits of
`this invention can comprise one or more containers and/or
`receptacles to hold the reagents of the kit, along with appro-
`priate buffers and/or diluents and directionsfor using the kit,
`as would be well knownin theart.
`
`The following examples are not intended to limit the scope
`of the claims to the invention, but are rather intended to be
`exemplary of certain embodiments. Anyvariations in the
`exemplified methods that occur to the skilled artisan are
`intendedto fall within the scope ofthe present invention. As
`will be understood by one skilled in the art, there are several
`embodiments and elements for each aspect of the claimed
`invention, and all combinations of different elements are
`hereby anticipated, so the specific combinations exemplified
`herein are not to be construed as limitations in the scope ofthe
`invention as claimed. If specific elements are removed or
`added to the group of elements available in a combination,
`then the group of elementsis to be construed as having incor-
`porated such a change.
`
`EXAMPLES
`
`Example 1
`
`Determination of Feasibility of Extracting Both
`Protein and DNA from a Single Sample Using a
`Dual Extraction Method
`
`Summary of Results.
`Several parameters were tested to optimize protein extrac-
`tion of maize leaf tissue followed by DNA extraction of the
`remaining tissue. Seventeen different trait assays were ana-
`lyzed and both lyophilized and frozen leaf tissue were tested.
`Logistics of testing large numbers of samples were explored.
`Processing the samples for both protein and DNA worked
`well for all of the traits tested. This process worked well on
`both frozen and lyophilized tissue and can be performed
`following current high-throughput methods.
`Methods.
`Newly transformed maize plants were sampledin separate
`blocks for both secondary TaqMan and ELISA following the
`standard sampling protocol. The samples in the secondary
`TaqManblock were processed normally. The samples in the
`ELISA block were ground using a tissue homogenizer(e.g.,
`Kleco or tissue pulverizer) and extracted for protein by the
`addition of0.25 ml PBST (phosphate buffered saline contain-
`ing 0.05% Tween-20) followed by manual shaking of the
`block. The block was spun for 5 min at 4000 rpm and 0.2 ml
`
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`US 8,927,214 B2
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`7
`of the supernatant containing nondenatured proteins was
`transferred to another block for subsequent ELISA process-
`ing andtesting.
`Onthe pellet, a modified DNA extraction procedure was
`performed. DNA lysis buffer (0.2 ml) was added and the
`block was shaken on the tissue homogenizer for 10 sec and
`then spun for 10 min. This supernatant containing the DNA
`wasthen processed according to a DNA isolation procedure
`using magbeads. Briefly, 0.1 ml of the supernatant is trans-
`ferred to a 96-well plate (e.g., multiwall plate) containing 5 pl
`per well of magbeads. The multiwell plate is positioned on a
`magnet for a period oftime sufficient for nucleic acid present
`in the supernatant to bind the magnetic beads in the wells of
`the multiwell plate. The supernatantfluid is removed from the
`wells of the multiwell plate and the magnetic beads are
`washedwith wash buffer [e.g., 50% PA wash buffer (125 mM
`Tris pH 7.5, 25 mM EDTA, 500 mM NaCl), 25% isopropanol
`and 25% ethanol (95%)]. The multiwall plate is again posi-
`tioned on the magnet and wash buffer is removed. The wash-
`ing and positioning steps are repeated at least two times. The
`magnetic beads are then dried under conditions whereby the
`ethanol in the wash buffer evaporates. Elution buffer [non-
`limiting examples of which include water, Promega elution
`buffer, low TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0), TE
`buffer (10 mM Tris, 1 mM EDTA, pH 8.0)] is then added to
`the wells of the multiwell plate to elute the nucleic acid from
`the magnetic beads. The multiwall plate is positioned on the
`magnetfor a period of timesufficient for the magnetic beads
`to collect at the bottom ofthe wells ofthe multiwell plate. The
`elution buffer containing the nucleic acid can be removed
`from the wells ofthe multiwell plate for further analysis ofthe
`nucleic acid and/orthe elution buffer containing the nucleic
`acid can remain in the wells of the multiwall plate and be
`contacted directly with reagents(e.g., reagents for amplifica-
`tion of nucleic acid) for further analysis of the nucleic acid.
`Results.
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`extraction of DNA from what remainedofthe single tissue
`sample. The results from this dual extraction method are
`comparable to the standard DNA extraction methodresults
`and ELISA results. Large numbers of samples were pro-
`cessed utilizing existing high through-put processes without
`affecting protein or DNA results. Implementing this dual
`extraction process could potentially reduce cost of analysis
`by 40-50%.
`
`Example 2
`
`Protein/DNA Dual Extraction
`
`Comparison ofDual Protein/DNA Extraction to a Standard
`DNA Extraction.
`
`Maize leaf tissue from 8 different plants was sampled in
`duplicate in 2 separate deep 96-well blocks. One block was
`processed by a standard DNA extraction methodandthe other
`block was extracted first for protein followed by the same
`standard DNA extraction method. The DNA recovered from
`
`both extractions was tested for two genesusing real time PCR
`(polymerase chain reaction) ‘laqMan technology, which can
`measure the numberofcopiesof a specific gene. The average
`Ct value of the endogenous gene (TET) was compared for
`each gene measured and was within the 22-26 acceptable
`range for both DNA extraction methods. The Ct value indi-
`cated that the quantity of the DNA extracted wassufficient to
`obtain good results. Copy calls can be determined by the
`groupingsin the scatter plots. Tightness of the groupings can
`be used as an indicator of the quality. The number of copies
`determined from the DNAextracted using the dual extraction
`methodis identical to the numberof copies determined from
`the standard DNA extraction method. Extracting protein first
`before isolating the DNA doesnotaffect the outcome of the
`real time PCRresults.
`
`Seventeen different TaqMan assays were used for this com-
`parison over a 4 monthtesting period.
`Additional testing was performed on a large field sample
`data set to explore the logistics of testing large numbers of
`samples. Existing instrumentation was used to process these
`samples following minimum changesto current high though-
`put processes. No difference was seenin data quality between
`samples sitting in protein extraction buffer from 15 min to 165
`min before the addition of DNA lysis buffer or in samples
`sitting in DNA extraction buffer for up to 24 hr before pro-
`cessing. Also, greater than 99% of the samples recovered
`sufficient DNA to obtain a quality result.
`This method would reduce the amount of sampling from 5
`blocks to 2 blocks (40% reduction) as well as lowerthe cost of
`consumables.
`Conclusions.
`
`Criteria used for determining best conditions includedthat
`the endogenousgene (TET) cycling time (Ct) value had to be
`within range (22-26) and 0.2 ml of protein extract with suf-
`ficient total protein was needed for ELISAtesting. Optimum
`leaf tissue size was determinedto be 4 leaf discs and the best
`
`protein extraction buffer was PBST.
`Several samples were analyzed following the dual extrac-
`tion method and compared to the results from samples
`extracted by a standard TaqMan DNAextraction method
`(e.g., Promega DNAextraction kit). No significantdifference
`in the results was seen between the two extraction methods.
`
`40
`
`45
`
`Quality of DNA from the Dual Extraction Method.
`To evaluate the quality of DNA obtained from the dual
`extraction method and the robustness of this method, over
`2500 maize leaf samples were tested in 17 different real time
`PCR (TaqMan) assays. Numberof copies of each gene, aver-
`age Ct value and DNA concentration were compared. The
`average TET Ctvalueforthe dual extraction method matched
`the corresponding standard DNA extraction method. Also,
`copy call matches between the two processes averaged
`greater than 93%. Average DNA concentrations recovered, as
`measured by Pico green, were very similar, indicating that
`extracting protein first does not prevent good DNArecovery.
`Testing Process in a High-Throughput Format.
`Over 2400 lyophilized maize leaf samples were tested
`using the dual protein/DNA extraction method. The protein
`extract was tested by ELISA and the extracted DNA was
`tested in 3 different TaqMan assays. Automation processes
`were employed in order to process this number of samples.
`During the processing, samples were in protein extraction
`buffer for 15 to 165 min before the addition of DNA lysis
`buffer to begin the DNA extraction. Greater than 98% of the
`samples were in agreement between the protein ELISA
`results and the DNA TaqMan results. Less than 1% of the
`samples resulted in a poor DNA extraction with no results
`obtained. The results also show that the DNA in planttissue
`extracted in protein extraction buffer is stable for at least 2.5
`A dual extraction method was developed that allowed the
`hours. No difference in average TET Ct value was seen
`isolation of both protein and DNAfromasingle planttissue
`between samples which remainedin protein extraction buffer
`sample. The protein was extractedfirst in orderto isolate the
`for 15 minutes vs. 165 minutes.
`protein in its native form (non-denatured), followed by
`
`

`

`US 8,927,214 B2
`
`9
`Example 3
`
`Stability Studies
`
`Stability of the leaf extract in the protein extraction buffer
`or the DNA lysis buffer was evaluated. Samples were incu-
`bated in PBST,pellet without PBST, or DNA lysis buffer for
`up to 3 days at room temperatureor at 4° C. before extracting
`the DNA.For ELISAtesting, mostof the proteins tested are
`stable for up to 72 hrs at 4° C. in PBST. Both lyophilized
`tissue and fresh tissue stability were equivalent. Room tem-
`perature storage showed more degradation over time and is
`not recommendedfor storing the extracts.
`For DNAstability, DNA was extracted from thetissue from
`each of the 3 storage conditions described above (in PBST,
`pellet without PBST, or DNAlysis buffer). DNA stability was
`evaluated by analyzing both the median endogenous cycle
`value (median TET Ct) and standard deviation ofthe samples.
`These two measures will give an indication of DNA quality
`and will determine the accuracy of the copy call. Thus as
`DNAstability/quality decreases, copy calls will be affected.
`DNAin extracts stored at 4° C. in PBST or without PBST
`were stable, with good TET Ct values and low standard devia-
`tions for both fresh and lyophilized tissue. Extracts stored in
`lysis buffer had higher standard deviationsat 24 hrs, indicat-
`ing a decrease in DNA quality. Room temperature storage
`experiments also had higher standard deviations.
`Resu