Volume 13 Number 20 1985
`
`Nucleic Acids Research
`
`Rapid transfer of DNA from agarose gels to nylon membranes
`
`Ken C.Reed and David A.Mann
`
`Department of Biochemistry, Faculty of Science, Australian National University, Canberra A.C.T.
`2601, Australia
`
`Received 3 September 1985; Accepted 26 September 1985
`
`ABSTRACT
`The unique properties of nylon membranes allow for dramatic improvement in the capillary
`transfer of DNA restriction fragments from agarose gels (Southem blotting). By using 0.4 M NaOH as the
`transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer.
`Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline
`solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by
`this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in
`sensitivity of subsequent hybridization analyses.
`In addition, we have found that nylon membrane
`completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including
`distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4
`Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from
`polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon
`membrane, reduces its ability to hybridize.
`
`INTRODUCTION
`The capillary transfer of DNA restriction fragments from an agarose gel to an appropriate
`membrane ('Southern blotting'; 1) is a technique fundamental to the analysis of genome organization
`and expression. In recent years, the availability of nylon membranes as matrices for binding nucleic acids
`has markedly enhanced the utility of both this and allied techniques in hybridization analyses. Relative
`to nitrocellulose, nylon membranes have greater mechanical strength, have a higher capacity for nucleic
`acids, are able to bind smaller oligonucleotides, and have stronger retention of bound nucleic acids
`permitting multiple re-probing of filters (refer to technical bulletins of suppliers). In addition, it has been
`reported recently that ultraviolet irradiation catalyzes the covalent attachment of nucleic acids to these
`membranes. They thus provide all the advantages of chemically-activated cellulose papers (2, 3) without
`any apparent disadvantages.
`Nevertheless, it seemed to us that the full potential of nylon membranes is not realized in transfer
`protocols that are currently in use. These owe more to their historical succession from Southern's
`studies with nitrocellulose (1) than to intrinsic properties of the membranes themselves. For example,
`the fact that nylon membranes can be used in electrophoretic transfer argues that they bind nucleic
`acids efficiently in buffers of low ionic strength, in marked contrast to nitrocellulose. The use of low-salt
`buffers for capillary transfer would be expected to improve transfer efficiency, and would certainly be
`more convenient (in this context we note that Amersham, the suppliers of 'Hybond N' nylon membrane,
`
`© I RL Press Limited, Oxford, England.
`
`7207
`
`Enzo Exhibit 2014
`BD v. Enzo
`Case IPR2017-00172
`
`Exhibit 2014 Page 1
`
`

`

`Nucleic Acids Research
`
`suggest the use of 25 mM phosphate for capillary transfer).
`Indirect observations in our laboratory had suggested further that nylon membranes may be able
`to bind native (double-stranded) DNA, in addition to denatured (single-stranded) DNA. The potential
`saving in time and materials afforded by elimination of gel pre-treatment steps prompted us to
`investigate this question in more detail.
`In the course of these studies we found that DNA is retained by nylon membrane when
`transferred in acid or alkaline solvents. Not only does this allow large fragments to be sheared by
`depurination during transfer, but alkaline solvents also promote covalent fixation of DNA to the
`membrane. The convenience of these direct transfer procedures is complemented by a marked
`improvement in the resolution and sensitivity of subsequent hybridization analyses.
`
`MATERIALS AND METHODS
`DNA Preparations.
`Bovine genomic DNA was isolated from homogenates of frozen liver samples prepared in 10
`volumes of ice-cold NTE (100 mM NaCI, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA); nuclei were lysed by the
`addition of EDTA to 0.1 M and sarcosyl (sodium N-lauroyl sarcosinate; Sigma) to 2% (w/v), followed by
`incubation at 650C for 15 min. Solid CsCI was added at 1 g/ml, the suspension was made 0.7 mg/ml in
`ethidium bromide and centrifuged at 45,000 rpm in a Beckman Ti8O rotor at 250C for 60 h. The band of
`DNA was removed by side puncture, ethidium bromide extracted with n-butanol, and the sample
`dialyzed exhaustively against TE (10 mM Tris-HCI (pH 8.0), 1 mM EDTA). The final DNA solution was
`stored at 40C over a few drops of chloroform.
`Human genomic DNA was isolated similarly from the Burkitt lymphoma-derived cell lines BJAB
`(female; provided by Dr. Barry Gorman, QIMR) and RAJI (male; ATCC).
`Plasmid pSPIB3.8 is pSP64 (4) containing a 3.8 Kb Bam Hi fragment of the human X
`chromosome (5). The 812 bp Eco RI/Bam Hi fragment of this plasmid, which contains the promoter and
`first intron of the X-linked gene for phosphoglycerate kinase, was sub-cloned by digestion of pSPIB3.8
`with Eco RI followed by re-circularization with T4 DNA ligase and transformation into HB101 (6). The
`resultant recombinant (pSPIBa.8) was amplified in liquid culture and the plasmid isolated and purified
`according to the method of Bimboim and Doly (7), with the inclusion of a final purification by isopycnic
`centrifugation in CsCI and ethidium bromide (8).
`Bacteriophage Lambda DNA (cl857Sam7) was obtained from New England BioLabs.
`Salmon sperm DNA (Sigma), used as the carrier in filter hybridizations, was dissolved in 0.2 M
`NaOH at 10 mg/ml and sheared by heating at 1000C for 45 min. The solution was then chilled,
`neutralized with acetic acid and centrifuged to remove debris. DNA was recovered by ethanol
`precipitation and the pellet dissolved in TE and stored in small aliquots at -200C.
`Restriction Endonuclease Digestions.
`Samples of DNA were digested with the appropriate restriction endonuclease(s), obtained
`variously from Bethesda Research Laboratories, New England BioLabs, Boehringer, and Pharmacia-PL,
`under conditions recommended by the suppliers.
`
`7208
`
`
`Exhibit 2014 Page 2
`
`

`

`Nucleic Acids Research
`
`End-Labelling of DNA Digested with Restriction Endonuclease.
`Bam Hi-digested genomic DNA was incubated with the large (Klenow) fragment of DNA
`polymerase I (New England BioLabs) with 0.2 mM dGTP and [a-32PJ-dATP (Amersham) at room
`temperature for 30 min (8).
`Agarose Gel Electrophoresis.
`Gels were cast and run in the Pharmacia GNA-1 00 mini-gel apparatus, using either 5- or 8-tooth
`combs that form sample wells of 10 mm x 1 mm x 7 mm and 4.5 mm x 1 mm x 7 mm respectively. The gel
`volume was 60 ml, usually of 1% (w/v) agarose (Sigma Type I) in TAE buffer (40 mM Tris-acetate (pH 7.8),
`20 mM sodium acetate, 2 mM EDTA) containing ethidium bromide (0.5 ±g/ml); the gel dimensions were
`100 x 80 x 7.5 mm. Electrophoresis was conducted at 125 mA/45 V (2.8 V/cm) for 100-200 min at room
`temperature.
`Gel Pre-Treatment.
`Following electrophoresis, each gel was photographed on a medium-wavelength (302 nm)
`ultraviolet transilluminator (Oliphant, Adelaide) then immediately subjected to one of the following
`pre-treatments (for treatments (A) and (B), the free-floating gel was agitated gently and continuously at
`room temperature):
`(A) Acid/alkalUneutralization (depurination): the gel was treated sequentially with 2 volumes of
`0.25 M HCI (2 x 10 min), 2 volumes of 0.5 M NaOH/1.5 M NaCI (2 x 15 min), and 2 volumes of 0.5 M
`Tris-HCI (pH 7.5)/1.5 M NaCI (2 x15 min) (9).
`(B) Add: the gel was treated with 2 volumes of 0.25 M HCI (2 x 10 min).
`(C) Ultraviolet irradiation: the gel was placed 1 cm below the germicidal ultraviolet strip light (254
`nm, 30 W) in a biosafety cabinet. Each side of the gel was exposed for half the total time indicated in the
`text, with the origin placed directly beneath the light to ensure that the largest DNA fragments received
`the greatest exposure (10).
`(D) Nlo pre-treatment.
`Capillary Transfer to Nylon Membranes.
`For overnight transfer, the pre-treated gel was placed on three sheets of saturated Whatman
`3MM paper supported on an inverted gel casting tray in a small plastic box. The paper was the same
`width as the tray and had extended wicks dipping into a reservoir of solvent in the bottom of the box. A
`sheet of Zeta Probe nylon membrane (Bio-Rad), cut to the same dimensions as the gel and previously
`wetted in distilled water, was placed on the gel surface and this in turn was covered with eight sheets of
`3MM paper and a stack of paper towel to the height of the box rim. Light pressure was applied to the
`stack by sealing the box with a snap-seal lid.
`For shorter transfer periods, the protocol adopted was similar to that described by Wahl et al. (9).
`To effect a change of solvent, a glass plate was placed on top of the transfer assembly and the assembly
`inverted. The pad containing transfer solvent was carefully peeled off the base of the gel and replaced
`with a fresh pad saturated with the new solvent, the assembly was returned to its original orientation and
`transfer resumed.
`
`7209
`
`
`Exhibit 2014 Page 3
`
`

`

`Nucleic Acids Research
`
`Fixation of Transferred DNA.
`Following the transfer of native DNA, the membrane was removed from the gel and placed, DNA
`surface uppermost, on a pad of 3MM paper saturated with 0.5 M NaOH/1.5 M NaCI for 5 min, then placed
`for 5 min on a second pad saturated with 0.5 M Tris-HCI (pH 7.5)/1.5 M NaCI (1 1).
`These membranes, together with those to which denatured DNA had been transferred, were
`agitated briefly in 2 x SSC (0.3 M NaCI, 0.03 M trisodium citrate) to remove possible adherent gel
`fragments and, in the case of alkaline transfers, to neutralize the membranes. They were then blotted
`dry and subjected to one of the following fixation procedures:
`(a) Baked in a vacuum oven at 800C for 2 h (1).
`(b) Wrapped in Glad Wrap (equivalent to Saran Wrap) and placed, DNA surface down, on the
`ultraviolet transilluminator (refer above) for the times specified.
`Placed, DNA surface uppermost, on a clean sheet of 3MM on the work surface of the
`(c)
`biosafety cabinet (refer above) and exposed to its ultraviolet light for the times specified. The light
`source was located 60 cm above the membrane.
`Nick-Translation of DNA.
`Probes for hybridization analyses (and, in the experiments of Figure 2, genomic DNA) were
`labelled with [a-32P]-dCTP (Amersham) according to the principle of Rigby et al. (12), using a
`modification that ensures high efficiency of label incorporation (13).
`Hybridization of DNA Bound to Nylon Membranes.
`In most cases, the hybridization solution consisted of 1.5 x SSPE (0.27 M NaCI, 15 mM sodium
`phosphate (pH 7.7), 1.5 mM EDTA), 0.5% (w/v) BLOTTO ('Diploma' non-fat powdered milk; 14), and 1%
`(w/v) SDS. To ensure uniformity of hybridization conditions, all membranes prepared for a single
`comparative experiment were placed in a small plastic box with 1 ml/4 cm2 of hybridization solution
`containing heat-denatured, sheared salmon sperm DNA (0.5 mg/ml). A sheet of Glad Wrap was molded
`to the inside, in contact with the surface of the solution and overhanging the edges of the box, and the
`Pre-hybridization was continued overnight at 680C with continuous
`box closed with a snap-seal lid.
`agitation.
`The radio-labelled probe was mixed with salmon sperm DNA in 0.2 M NaOH, heated at 1 000C for
`10 min, chilled, and neutralized with acetic acid. The solution of denatured, sheared probe and carrier
`DNA (final concentration 0.5 mg/ml) was mixed with fresh hybridization solution (1 ml/8 cm2 of
`membrane), the membranes were added and hybridization continued at 680C for up to 20 h.
`Subsequent studies have shown that the omission of carrier DNA from prehybridization and
`hybridization solutions has no adverse effects on either background or sensitivity under these
`conditions.
`When probing for single-copy fragments of the PGK gene, prehybridization was carried out at
`420C in plastic bags containing 10 ml of 4 x SSPE, 0.5% BLOTTO, 1% SDS, and 0.5 mg/ml salmon
`Hybridization was performed under similar conditions, with the
`sperm DNA in 50% (v/v) formamide.
`It was found subsequently that in using dextran sulfate, carrier
`inclusion of 10% (w/v) dextran sulfate (9).
`
`7210
`
`
`Exhibit 2014 Page 4
`
`

`

`Nucleic Acids Research
`
`DNA could be omitted from hybridization solutions but not from the prehybridizations.
`Washing of Membranes and Autoradiography.
`On completion of hybridization, the membranes were rinsed briefly in 2 x SSC/0.1% SDS then
`washed successively (for 15 min at room temperature with vigorous agitation) in 2 x SSC/0.1% SDS, 0.5
`x SSC/0.1% SDS and 0.1 x SSC/0.1% SDS, with a final high-stringency wash in 0.1 x SSC/1% SDS at
`5000 for 30 min. The membranes were blotted dry, wrapped in Glad Wrap and exposed to pre-flashed
`X-ray film (Fuji RX) with an intensifying screen (DuPont Cronex 'Lightning Plus') at -700C (15).
`Fixation and Drying of Gels.
`On completion of transfer of radio-labelled restriction fragments, the gels were soaked in 7% (w/v)
`trichloroacetic acid for 30 min then dried by blotting with 3MM paper under a moderate weight (8). The
`dried gels and the membranes used in transfer were wrapped in Glad Wrap and autoradiographed at
`room temperature.
`
`RESULTS
`Retention of Native DNA by Nylon Membrane.
`Initial expenments were designed simply to determine (i) whether Zeta Probe retains DNA at lower
`ionic strength than is recommended in standard protocols, and (ii) whether it binds native DNA with an
`efficiency comparable to that found with denatured DNA.
`Each of the 8 tracks of a gel contained
`
`A
`_
`
`3.0
`0.8_
`
`C
`3.0 E Iu4--_
`0.8
`_
`
`I>___
`
`B
`
`DR
`
`Figure 1. Transfer of native DNA to Zeta Probe. Bovine genomic DNA (0.8 gg, digested with Eco RI)
`was mixed with doubling dilutions of pSPIBO.8 (6.4-0.05 ng, digested with Bam Hi and Eco RI) and
`electrophoresed on 1% agarose gels. The resolved fragments were transferred to Zeta Probe
`overnight, either directly ((c), (d)) or after first being subjected to acid depurination ((a), (b)); the transfer
`solvent was either 1 x SSC ((a), (c)) or 10 x SSC ((b), (d)). The membranes were rinsed (in (c) and (d),
`following denaturation), baked, and hybridized with nick-translated pSPIBO.8 (30 jLCVi/g at 25 ng/ml) for
`20 h, then washed and autoradiographed overnight. Numbers at the left refer to sizes (Kb) of the vector
`and insert fragments .
`
`7211
`
`
`Exhibit 2014 Page 5
`
`

`

`Nucleic Acids Research
`
`identical amounts of bovine genomic DNA (0.8 ,g, digested with Eco RI), together with doubling
`dilutions of pSPIB0.8 (6.4-0.05 ng, digested with Bam HI and Eco RI) as the target for hybridization
`analysis.
`The transfer and retention of depurinated DNA (treatment (A), Materials and Methods) in 1 x SSC
`is at least equal to that in 10 x SSC, the recommended solvent (Figs. la, lb); in fact, the lower ionic
`strength results in a two-fold stronger hybridization signal, implying more efficient transfer. Of greater
`interest is the comparison of results obtained with the transfer of depurinated and native DNA in 1 x SSC
`(Figs. la, 1c). Clearly, native DNA fragments of the sizes detected (3.0 and 0.8 Kb) are transferred and
`bound with similar efficiency to sheared, denatured fragments. Furthermore, the bands of hybridized
`DNA are less diffuse, an expected consequence of eliminating the pre-treatment steps.
`The hybridization signals detected following transfer of native DNA in 10 x SSC are markedly
`weaker than those seen with 1 x SSC (Figs. 1d, 1c). This probably reflects their less efficient transfer at
`high ionic strength, since the signal intensity for the 812 bp insert is similar to that for the 3 Kb vector
`suggesting poorer elution of larger fragments.
`Extension of these studies has shown that electrophoretic transfer of native DNA fragments from
`polyacrylamide gels to Zeta Probe similarly occurs at high efficiency and with improved resolution and
`sensitivity (Klaus Matthaei, unpublished observations).
`In Situ Fragmentation of Native DNA by Ultraviolet Light.
`While direct capillary transfer of native DNA is useful for screening relatively small DNA molecules
`(eg. recombinant plasmids digested with restriction enzymes), it is less than quantitative for fragments
`larger than 4 Kb (cf. 1, 9). We therefore used short-wavelength ultraviolet irradiation in the presence of
`ethidium bromide to facilitate the transfer of larger molecules by fragmenting native DNA within the gel
`(10). Exposure to the light source in our biosafety cabinet for 20 min (treatment (C)) was necessary and
`sufficient to ensure that radio-labelled DNA from all regions of the gel transferred at a rate and efficiency
`similar to that obtained with depurination (eg. Figure 2; however, see also Figure 4).
`Effect of Ionic Strength on Elution and Retention.
`This treatment allowed a more detailed examination of the effect of ionic strength on the elution
`and retention of both native and denatured fragments. For these studies, we used radio-labelled DNA
`fragments generated by digestion of nick-translated bovine genomic DNA with Alu I, Hae IlIl (mean
`fragment sizes - 250 bp), Eco RI and Bam HI (mean fragment sizes - 4200 bp), together with
`undigested DNA. Following electrophoresis of the samples in eight replicate gels, four of the gels were
`subjected to ultraviolet irradiation (treatment (C); native DNA) and four were subjected to depurination
`(treatment (A); denatured DNA) to fragment large molecules. DNA was then transferred for 3 h in
`distilled water, 1 x SSC, 6 x SSC, or 20 x SSC.
`Autoradiographs of the gels and membranes revealed a slight but noticeable decrease in transfer
`with increasing salt concentration for both native and denatured DNA (data not included).
`However,
`retention of DNA by the membranes was quite independent of ionic strength, illustrated clearly by
`Figure 2 which shows the results from the transfer of native and denatured DNA in distilled water. The
`
`7212
`
`
`Exhibit 2014 Page 6
`
`

`

`Nucleic Acids Research
`
`.e;.~
`
`Seal
`
`1 2 345 54 32 1
`
`FigureZ.
`Transfer and retention of fragmented native and denatured DNA in distilled water. Bovine
`genomic DNA was Labelled by nick-translation to 0.2 j±CVpg; 1 pLg samples were digested with restriction
`enzymes as described and electrophoresed in 1% agarose gels. Replicate gels were subjected to acid
`depurination (b) or exposed to high-intensity ultraviolet irradiation (c), and DNA transferred to Zeta Probe
`for 3 h in distilled water. The membranes and dried gels were autoradiographed for 2 h at room
`temperature, together with an untreated (control) gel (a). Restriction enzymes used were: lane 1, none;
`lane 2, Alu I; lane 3, 8am HI; lane 4, Eco Rl; lane 5, Hae Ill. In each case, the autoradiograph of the dried
`gel is shown on the left, the filter on the right. Marks indicate the position of bromiophenol blue tracking
`dye.
`
`7213
`
`
`Exhibit 2014 Page 7
`
`

`

`Nucleic Acids Research
`
`A
`3.0 in __-
`m
`0.8
`
`B
`_
`
`D
`
`0
`
`C
`la
`-
`
`A
`
`3.0
`0.8
`
`--
`
`_
`
`Figure 3.
`Fixation of transferred DNA to Zeta Probe. DNA samples were electrophoresed in 1%
`agarose gels as described for Figure 1.
`All gels were subjected to acid depurination and the DNA
`transferred ovemight to Zeta Probe in 10 x SSC. The membranes were rinsed in 2 x SSC and subjected
`to the following fixation procedures: (a) transilluminator (302 nm u.v.) for 1 min; (b) transilluminator for 5
`min; (c) biohazard cabinet (254 nm u.v.) for I min; (d) biohazard cabinet for 5 min. Hybridization and
`autoradiography were as described for Figure 1. Numbers at the left refer to sizes (Kb) of the vector and
`insert fragments.
`
`high level of retention of fragments from all regions of the gels (i.e. all size classes) was confirmed
`qualitatively by our finding that in all cases, the stack of absorbent paper above the membrane contained
`barely-detectable levels of radioactivity. This observation was common to all our experiments with
`radio-labelled fragments, regardless of the transfer solvent used.
`Additional studies have shown that glyoxalated RNA (16, 17) also is transferred and retained
`efficiently in distilled water (data not included).
`Post-Transfer Fixation of DNA to Zeta Probe.
`Before undertaking more detailed hybridization analyses, we compared the efficiency of
`altemative procedures for fixation of DNA to the membranes after transfer. The experimental design was
`similar to that of Fig. lb. Exposure of the membrane for 1 min to the medium-wavelength ultraviolet
`irradiation of the transilluminator (Fig. 3a) or the short-wavelength irradiation of the source in the
`biosafety cabinet (Fig. 3c) was clearly inadequate. However, 5 min irradiation with either light source
`(Figs. 3b, 3d) was equivalent to (or better than) vacuum baking for 2 h (Fig. 1 b).
`Consequently, in all following experiments involving hybridization, denatured DNA was fixed to
`the membranes after transfer (and denaturation where appropriate) by 5 min irradiation in the biosafety
`cabinet. This is more efficient than vacuum baking (it is certainly much faster), and the use of an
`unfiltered light source avoids solarization of the expensive quartz filters on transilluminators (cf. 9).
`
`7214
`
`
`Exhibit 2014 Page 8
`
`

`

`Nucleic Acids Research
`
`B
`
`A
`
`32.7-
`1 .3r
`
`0.5 -
`
`Ultraviolet shearing prevents hybridization. Bovine genomic DNA (0.8 jg, digested with Eco
`Figure 4.
`RI) was mixed with doubling dilutions of bacteriophage lambda DNA (6.4-0.05 ng, digested with Sal I)
`The resolved fragments were subjected to acid
`and electrophoresed on 0.6% agarose gels.
`depurination (a) or high-intensity ultraviolet irradiation for 20 min (b), then transferred to Zeta Probe for 3
`h in distilled water. The filters were rinsed (in (b), following denaturation), exposed to low-intensity u.v.
`for 5 min, hybridized with nick-translated lambda DNA (50 gCi/jg at 20 ng/ml) for 16 h, washed and
`autoradiographed. Numbers at the left refer to sizes (Kb) of the lambda DNA fragments.
`
`Ultraviolet Irradiation Inhibits Hybridization.
`Using optimal parameters established in the preceding studies, a Southern blot was prepared to
`determine whether high-intensity u.v. irradiation in the presence of ethidium bromide has deleterious
`effects on the hybridization of transferred DNA. The experimental design was similar to that described in
`Fig. 1, with the exceptions that the target for hybridization was the large fragments generated by
`digestion of lambda DNA with Sal I, and native DNA was fragmented by exposure to ultraviolet light
`Figure 4 compares these results with those obtained from an otherwise identical
`(treatment (C)).
`experiment in which denatured fragments were transferred after depurination (treatment (A)).
`High-intensity ultraviolet irradiation causes a dramatic loss of sensitivity (Figure 4), of the order of
`40-fold at the intensity and duration that is required to break large molecules to a size suitable for
`quantitative transfer. This treatment obviously induces structural alterations that prevent DNA from
`forming stable hybrids; by implication, these findings impose a serious limitation on the use of ultraviolet
`light for post-transfer fixation.
`Depurination by Transfer in Alkali.
`Since acid depurination remains the method of choice for fragmenting large DNA molecules
`within gels, we sought to determine if the DNA-binding properties of Zeta Probe would allow a rapid
`modification of this procedure. Two alternatives were considered: (i) pre-treatment of the gel in acid
`(treatment (B)) followed by transfer in 0.25 M NaOH; (ii) no pre-treatment, using 0.25 M HCI as the
`transfer solvent for the first 30 min, followed by transfer in 0.25 M NaOH for a further 150 min.
`Elution and retention were moniored with end-labelled fragments of bovine and human genomic
`DNA digested with Bam HI. After 3 h transfer, >98% of the radioactivity was bound to the membranes in
`both cases (Figure 5). Differences between treatments in the amount and distribution of radioactivity
`
`7215
`
`
`Exhibit 2014 Page 9
`
`

`

`Nucleic Acids Research
`
`A
`
`B_
`
`1 2
`
`3 4
`
`4 3
`
`2 1
`
`Transfer and retention of DNA in alkali. Samples (4 jg) of genomic DNA from human male
`Figure 5.
`(lane 1) and female (lane 2) cell lines, and bovine male (lane 3) and female (lane 4) liver were digested
`with Bam Hi, end-labelled to 0.5 jCVg±g, and electrophoresed on 1% agarose gels. DNA was either
`transferred directly to Zeta Probe in 0.25 M HCI for 30 min followed by a change of transfer solvent to
`0.25 M NaOH for 150 min (a), or first pre-treated with 0.25 M HCI then rinsed briefly in water and
`transferred to Zeta Probe in 0.25 M NaOH for 3 h (b). In each case, autoradiographs of the dried gels are
`shown on the left and transfer membranes on the right. Marks indicate the position of bromophenol
`blue tracking dye.
`
`transferred to the membranes were minor; these were subsequently eliminated by the substitution of
`0.4 M for 0.25 M NaOH as the transfer solvent (see below). The autoradiographs ot Fig. 5 provide
`particularly striking evidence of the high resolution attainable with rapid transfer protocols: minor satellite
`bands are clearly resolved, despite being indistinct in the original gel photographs (not included).
`
`7216
`
`
`Exhibit 2014 Page 10
`
`

`

`Nucleic Acids Research
`
`C
`
`A
`
`B
`
`3.8-
`
`1 2 3 45 1 2345
`
`Figure 6. Detection of single-copy sequences in mammalian DNA following rapid transfer in alkali.
`Samples (4 jig) of genomic DNA from human male (lane 1) and female (lane 2) cell lines, and bovine male
`(lane 4) and female (lane 5) liver were digested with Bam HI and electrophoresed on 1% agarose gels,
`together with size markers of lambda DNA digested with Acc I (lane 3). After photography (a), DNA was
`either depurinated and transferred to Zeta Probe in distilled water for 3 h (b), or was transferred directly
`to Zeta Probe in 0.25 M HCI for 30 min followed by a change of transfer solvent to 0.25 M NaOH for 150
`min (c). The membranes were rinsed briefly in 2 x SSC and exposed to low-intensity u.v. irradiation for 5
`min, then hybridized in dextran sulphate with nick-translated pSPIBO.8 (90 jCVijg at 16 ng/ml), washed
`and autoradiographed overnight. The 3.8 Kb fragment derived from the human X chromosome is
`indicated.
`
`7217
`
`
`Exhibit 2014 Page 11
`
`

`

`Nucleic Acids Research
`
`TRANSFER WITH DEPURINATON
`
`TRANSFER OF NATIVE DNA
`
`1.
`
`2.
`
`3.
`
`Soak gel In 2 vol 0.25M HCI
`with gentle agitation until BPB
`changes color (5 -15 min).
`
`Rinse briefly In water;
`pre-wet nylon membrane
`In water.
`
`Transfer to membrane
`with 0.4M NaOH
`(2 h to overnight).
`
`4.
`
`Rinse membrane In 2 x SSC.
`
`1.
`
`2.
`
`3.
`
`4.
`
`Transfer directly to nylon
`membrane with water
`(2 h to overnight).
`
`Place membrane (DNA surface
`uppermost) on pad satd. with
`0.4M NaOH for 5 min.
`
`Rinse membrane In 2 x SSC.
`
`Expose membrane (DNA surface
`uppermost) to u.v. light In
`blohazard cabinet for 5 min.
`
`Prehybrldize and hybridize as required.
`
`Figure Z. Rapid transfer protocols.
`
`Southern Blots Prepared by Alkaline Transfer.
`The rapid depurination/transfer protocols were tested by preparing Southern blots from gels
`containing Bam HI-digested genomic DNA from human and bovine sources, both male and female. Two
`identical gels were subjected to the above treatments while a third underwent standard acid
`depurination followed by transfer in water. Nick-translated pSPIBO.8 was used to probe for X-linked PGK
`fragments.
`Autoradiographs of the membranes after hybridization showed a marked increase in sensitivity
`and resolution with both of the rapid procedures (Figure 6; data for acid pretreatment/alkaline transfer
`not included). This is seen clearly in a comparison of the signals generated by hybridization of the probe
`to the 3.8 Kb fragment of the human X chromosome (5):
`the band detected in these overnight
`exposures of male DNA includes less than 0.5 pg of the 812 bp probe sequence. The increased
`sensitivity is apparent also in the detection of additional fragments in both human and bovine DNA,
`presumably representing sequences with partial homology.
`The improved resolution is to be expected; the improved sensitivity is probably due to the fact
`that in this procedure the DNA is totally denatured when fixed to the membrane. In contrast, the
`standard procedure (treatment (A)) involves neutralization of denatured DNA at high salt concentration
`followed by transfer under neutral conditions, during both of which some renaturation may occur.
`We have found that if adequate depurination is to be achieved by sequential transfer in acid and
`alkali, the time required for preliminary transfer in acid (or more correctly, the volume of acid required for
`elution) is dependent on the strength of the electrophoresis buffer.
`For example, with TBE
`(Tris-borate-EDTA) up to 1 h is required. In this case, we find it advisable to use either a thick pad of
`acid-saturated 3MM paper (8 sheets) or wicks extending into a small solvent reservoir.
`In any event,
`
`7218
`
`
`Exhibit 2014 Page 12
`
`

`

`Nucleic Acids Research
`
`color change of the bromophenol blue tracking dye is a reliable index of acidification.
`Additional work has shown that the transfer of large fragments is more rapid in 0.4 M NaOH than in
`0.25 M NaOH, reflecting the need for high pH following acid treatment in order to achieve satisfactory
`depurination. Neither the retention of DNA by Zeta Probe nor hybridization sensitivity are diminished
`with the stronger alkali, even following continuous exposure during overnight transfer (data not
`included).
`A surprising bonus afforded by alkaline transfer is that it promotes the covalent fixation of
`transferred DNA to nylon membranes. We have found that radio-labelled DNA transferred in alkali
`cannot be stripped from the membrane (data not included). Furthermore, signal intensities generated
`by hybridization to fragments transferred in alkali were more than twice as strong as those from identical
`samples which had been additionally subjected for 5 min to the ultraviolet light of the biohazard cabinet
`(experiment similar to Fig. 4; data not included). Apparently this mild exposure to ultraviolet radiation,
`the minimum required for fixation of DNA transferred in neutral solvents, is sufficient to render half of the
`transferred DNA incapable of forming hybrids.
`
`DISCUSSION
`The properties of Zeta Probe allow virtually any modification of standard protocols for the capillary
`transfer of DNA from agarose gels for hybridization analysis. Solvents that result in quantitative retention
`include distilled water, 0.25 M HCI and 0.4 M NaOH, in addition to the concentrated buffers that have
`been used traditionally with nitrocellulose.
`Optimal transfer protocols are those in which pre-treatment of the gel is minimized (to limit
`diffusion and hence improve resolution) and the DNA is denatured immediately before fixation (to
`improve sensitivity), either by transfer in alkali or by post-transfer denaturation. We have adopted a
`routine procedure of acid pre-treatment followed by transfer in 0.4 M NaOH (Fig. 7).
`With alkaline transfer, the need for separate fixation treatments is eliminated since the solvent
`itself promotes base-catalyzed fixation of DNA to the membranes. The use of ultraviolet irradiation in this
`context is counter-productive since it diminishes the ability of fixed DNA to form hybrids.
`The sensitivity of hybridization analyses is increased approximately ten-fold by the rapidity,
`complete denaturation, and optimal fixation of the alkaline transfer protocols. Single-copy sequences
`are easily detected by hybridization to a Southern blot prepared from less than 1 ig of mammalian
`genomic DNA with an overnight exposure of X-ray film.
`If alkali is not used, we suggest distilled water (or a low concentration of a suitable buffer) as the
`transfer solvent for either of native (Fig. 7) or depurinated DNA. The inverse relation between ionic
`strength and transfer efficiency is negligible for small molecules, but is very pronounced for fragments
`larger than a few hundred bases (Fig. 1). Even at low salt concentrations, molecules larger than 4 Kb are
`not eluted quantitatively; high-intensity ultraviolet irradiation is unsatisfactory for shearing larger native
`fragments since it causes a dramatic reduction in hybridization efficiency. Similar considerations apply to
`the transfer of RNA for the preparation of 'Northem' blots.
`
`7219
`
`
`Exhibit 2014 Page 13
`
`

`

`Nucleic Acids Research
`
`On completion of transfer of native DNA, the membrane