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The use of heparin as a simple cost-effective means of controlling background In nudeic acid hybridization procedures

The use of heparin as a simple cost-effective means of controlling background In nudeic acid... Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Volume 12 Number 14 1984 Nucleic Acid s Research The use of faeparln as a simple cost-effective means of controlligg background in nucleic add hybridization procedures L.Singh and K.W.Jones Institute of Animal Genetics, University of Edinburgh, Edinburgh EH9 3JN, UK Received 16 May 1984; Revised and Accepted 3 July 1984 ABSTR/CT The use of heparin as a simple, effective and cheap substitute for conventional methods of controlling background in hybridization procedures is described and illustrated with reference to the use of DNA probes in filter and In s_itu hybridization. Possible specific mechanisms for this heparin effect are discussed. INTRODUCTION Refractory background radioactivity in nucleic acid filter hybridization varies in severity with different types of probes, annealing conditions and filte r membranes. Background problems are also encountered in i n fiiili hybridization where they are possibly even more intractable because of the unavoidable complexity in the substrate and the fact that vigorous non- specific methods of control, such as Denhardt's (1) solution cannot be used without damage to the cytological structure. The possibility of a relatively specific means of background control in in situ hybridization was suggested by Maitland et al (2). These workers described non-hybridization binding of labeled DMA probes which was specific to certain cell types. Such binding was substantially reduced by the inclusion in the annealing reaction of the polyanionic glycan, heparin but the optimal conditions for this effect were not determined. Heparin has a substrate binding affinity similar to that of DNA, especially with respect to certain proteins (3). Furthermore, it can abolish the binding of proteins which increase the affinity of DNA for nitrocellulose membranes by specificall y combining with them (4,5). Given this well-characterised property, it is surprising that the possible use of heparin controlling non-specific substrate binding of DNA in both filter and in in situ hybridization apparently has not been explored previously. Accordingly we have examined this aspect and have shown that the inclusion of heparin in the hybridization annealing solution alone can dispense entirely with the use of Denhardt's solution and carrier DNA- This modification result s in the consistent and complete abolition of background in both filte r and i n situ hybridization under a variety of different conditions. Moreover the modified hybridization protocol is much simpler, faster and © IRL Press Limited, Oxford, England. 5627 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research alBO considerably cheaper than existin g methods. MATERIALS AND METHODS DNA extraction . DNA from liver s of male and female mice was isolated by homogenizing the tissu e in TES buffer (3OraH Tris , 5mM EDTA, 50mM NaCl, pH.8.O), lysing the cell s with sarkosyl NL3O (Geigy) and by isopycnic centrifugation in CsCl- ethidium bromide gradients as previously described (6). DNA restrictio n and gel electrophoresis. Restrictio n enzymes and DNA polymerase were obtained from Boehringer Mannheim, New England Biolabs and Bethesda Research Laboratories. DNA was digested by incubating at a ratio of l^ig DNA t o 2 units of enzyme for 4-5h in the buffers and conditions specified by the manufacturer. 0.6 to 1% agarose gel s were run in a vertica l floating gel apparatus at 30V for ll-12h in a buffer containing 15raM Tris, 18mM NaH PO and 0.5mM EDTA, pH 7.8. 2 4 R1p<-+rophoretic transfer of nucleic acids. Electrophoretic transfer of gel-fractionated DNA t o nitrocellulose sheets (7) was carrie d out using a Hoeffer transfer unit after depurination (8). DNA restrictio n fragments were transferred from 2.5 mm thick agarose slab gels by th e following procedure: the ge l was gently shaken for 15 min in 250 ml of O.25M HC1, washed gently with distilled water to remove the residual acid, denatured in two changes 15 min each of 250 ml 0.2M NaOH and 0.6H NaCl and neutralize d in 3 changes each of 500 ml of transfer buffer (TB; O.O25H Na2HPO j/NaH2PO4, pH 6.5). The transfer was carried out in TB a t room temperatur e for lh at 27V keeping the unit cool with circulating cold tap water . The efficiency of transfer was assessed comparatively using the following membranes: Schleicher and Schull membrane filters RefJto. 402097, pore siz e 0.1 um; Gene Screen hybridizatio n transfer membrane Cat.No. NEF- 972, Lot No. 354 GS21; Pall Biodyne transfer membranes P/N BNNG3R, rating 1.2 micron, Lot No. 172340. After transfer, the membranes were carefully washed with TB t o remove residual agarose, dried at room temperature and baked at 8O°C for 2-3 h. Blot hybridization. The following stock solutions were prepared: 20x SET (0.5M NaCl, 0.03H Tris, 2mM EDTA, pH 7.4); 5O% W/V sodium dextran sulphate aqueous solution, stored a t 4-5°C; 50 mg/ml heparin sodium sal t grade II from porcine intestinal mucosa (Sigma,H-7OO5) in 4xSET, stored at 4-5°C for several months; 20% W/V sodium pyrophosphate; 2O% W/V sodium dodecyl sulphate (SDS). i) Hybridization without dpxtran sulphate: The filte r was firs t soaked in 4xSET and placed in a plasti c bag with 10ml of hybridization solution per 10xl4cm membrane. Hybridization salt solution contained 4xSET, 0.1% sodium pyrophosphate solution, 0^% 33S, 50 pq/ml heparin. To avoid the formation of ai r bubbles the solution was preheated to 65°C before use. After heat sealing, the bag was placed in a shaking distilled water bath and incubated with vigorous shaking at 65°C for 2-3h. During thi s time the hybridization 5628 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research bag was kept submerged by another water-filled plastic bag. The denatured, or single-strande d probe was then added to the bag without introducing air bubbles. The bag was then resealed and the solution throughly mixed. The bag was returned to the water bath at 65°t and incubation with vigorous shaking continued for 4-15h depending on the desired C T. For convenience, incubation was normally continued overnight. Ai l Hybridization in the presence of dextran Bulphai-e. The procedure is basically the same as described above except that the solution contained 10% dextran sulphate and lOx the concentration of heparin (500jig/ml). iii ) Washing membrane filters . After either hybridization procedure, the filte r was removed from the hybridization bag, briefl y dipped in 2xSET a t room temperature, re-sealed in a fresh plastic bag with 40O-50Oml of prewarraed wash buffer (2xSET,(X2% SDS) and vigorously agitated for 30min at 65°C The wash buffer was replaced with prewarmed fresh buffer and the step repeated. In the third, and final, washing the wash buffer was replaced with prewarmed buffer adjusted in SET concentration so as to achieve the desired stringency , in the work described here lxSET, 0.2% SDS, and agitated as before. Finally, the membrane was rinsed in 2xSET at room temperature, air drie d and autoradiographed. Filter s can be re-used after removal of the hybridized probe by washing in 0.1% wash buffer (lx wash buffer contains 50mM Tris-HCl, pH 8.0, 2raM EDTA, 0.5% sodium pyrophosphate) for 2h at 65°C Double-stranded DNA was labeled by nick-translation according to the method of Maniatis et al.(9), with minor modifications, using 32P-dCTP (sj«ct. 410 Ci/mM,) or 32P-dATP (sp^ct. 3000 Ci/raM, Padicchemical Centre, Amersham). The reactio n was stopped by adding 5>il of 0.5H EDTA (in the tota l incubation volume of 20ul). 10-20ug of E.coli carrier DNA was then added and the DNA precipitate d with 60>il of absolute ethanol (2 x volume) at -20°C for 2h or -70°C for 30min. Unincorporated 32P dCTP was removed by 3-4 washes in absolut e ethanol in a microcentrifuge. Finally, the precipitate was lyophilised , dissolved in 0.2ml of O.lxSET, denatured for 20min in boiling water , cooled rapidly on ice and used immediately or stored at -20°C. Occasionally, we have used a nick-translated probe without first removing the unincorporated 32P dCTP without experiencing any autoradiographic background problens. Preparation of single-stranded DNA probes. Single stranded DNA probes were prepared according to the method of Hu and Messing (10). A 16 base primer CACAATTCXaCACAAC complementary to the region 5" to the multiple cloning sites of M13rap7, M13mp6 and M13mp9 (New England Biolabs.) was used to initiate limited downstream synthesis of part of the complementary strand of the M13 sequence, leaving the cloned insert single- stranded- This partially single stranded probe labeled with 32P dATP (sp^ict. 3000 Ci/mM) had a specific activity of 3X1O8 cpm/ug. CvtQ.1onjc.a.J preparations. Microscope slide preparations were made from highly concentrated drops of mouse spermatozoa recovered by squeezing the dissected vas deferens in 0.9% 5629 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research NaCl solution. In some samples, the sperm were treated with hypotonic O.O75M KC1 solution for 8 min at 37°C Shears were fixed in 3:1 methanol-acetic acid for 30 min, air dried and stored at 4°C In situ hybr^d.jzation. To make the sperm nuclei accessible to the probe, the slides were incubated i n 50mH dithiothreito l (DTD in 0.1M Tris-HCl buffer, pH 8.0 at room temperatur e for 20 min., rinsed in 2xSSC (SSC i s 0.15M NaCl, 0.015M sodium citrate , pH 7.0), dehydrated through an (50%-100%) alcohol series and air dried. To remove histone and non-histone proteins, the slides were immersed i n a solution containing dextran sulphate (2rag/ml, Pharmacia), heparin (C2mg/ral, Sigma), lOraH Tris (pH 9.0), lOmM H7TA and OJ.% Nonidet P-40 at 5°C for 45 min, rinsed through two 15 min changes of 2xSSC , dehydrated through an ethanol series ,air dried and heat denatured (6). Air dried slide s were rinse d briefl y in heparin solution (50ug/ml in 1^0) and ai r dried without further rinsing. The nick translated DNA probe was denatured, lyophilised and diluted to the desired volume and salt concentration immediately before use. The hybridization mix contained nick-translated DNA (uncloned Bkm; Bkm clone 2(8), or lambda phage DNA), 1.7 ug/ml, sp.act. between 2xlO7 and 4xlO7 cpm/ug; 3xSSC; 10% dextran sulphate and 500 ug/ml heparin. No carrie r DNA was used. Where dextran sulphate was not used the concentration of heparin was reduced to 50 ug/ml. For in situ hybridization the general procedure of Jones (11) was followed with minor modifications. 6 ul of hybridization mix was used on each slide and annealing was carried out at the Tj^ . (60°C) for 4 hr. The hybridized slides were washed in 2xSSC at 60°C for In., washed overnight in 2xSSC a t 4°C, passed through an ethanol series and ai r dried. Hybridized slide s were dipped in Ilford K2 nuclear emulsion, exposed for 1 to 10 weeks, developed in Kodak Dl9b developer, fixed in Ilford Hypara, stained in Giemsa and photographed as described previously (12). RESITr.TS- Blot hybridization. Heparin concentrations over the range of 5 t o 10OOpg/ml were teste d in order to establish the minimum concentration effective in abolishing background in the absence of other measures. As might be expected, the higher the effective concentration and the specific activity of the probe the more hepari n needed to effectively supress background.At the probe specific activitie s achieved (5xlO7-3xlO^ cpm/ug),and with added probe of from 2xlO5-2xlO6 cpm/ml,it was found necessary to add 5O-200 jjg/ml of heparin in the absence of dextran sulphate. This was required to be increased to 500-700 .ug/ml of heparin in the presence of dextran sulphate. The tenfold higher concentration presumably is necessary to offset the concentrating effect of dextran sulphate on the probe. To obtain complete control of background it was found effective to include heparin only in the hybridization mix. Hybridization in dextran sulphate of the single-stranded cloned Bkm probe 2(8) (13) to an Alul digested mouse DNA blo t is shown in Fig 1. with and 5630 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research o CT ^ C Figure 1. (yjl of male and female mouse genomic DNA digeste d with Alul, fractionated on 1% agarose gel, transferred onto Schleicher & Schull membrane filter s and hybridized with, the -"P single-stranded probe 2(8) 6xlCP cpra/ml (specifi c activity 3.2x10° cpm/ug) for 12h at 65°C (see Experimental Procedures) exposed for 15h without pre-sensitizing the film. Note absence of background in panel 1 which was hybridized in the presence of dextran sulphat e and heparin. Panel 2 shows the same sequences hybridized under identical conditions in the absence of heparin. Note patchy background. without 50Qug/ml of heparin (panels 1 and 2 respectively). Heavy patchy background occurs only in the filte r hybridized in the absence of heparin. Hybridization to low copy number sequences in mouse and yeast genomic DNA using the heparin/dextran sulphate method is shown in Fig.2 . Panel 1 shows EcoRl digested male and female mouse DNA hybridized with a probe of a mouse male-specifi c fragment (H34) recovered from a mouse genomal library. The absence of background is evident from the female DNA track which lacks the sequence in question. Panel 2 shows yeas t DNA hybridize d with a cloned MAT sequence probe (2DX) and is , similarly, free from non-specific background. A brie f comparative assessment of the heparin and Denhardt methods was made 5631 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research er o. M34 2DX Figure 2. Hybridization of cloned probes of single copy sequences to genomic DNA i n the presence of heparin and dextran sulphate snowing absence of background.Panel 1: 6>jg of EcoRl digested male and female jnouse DNA transferred and hybridized for 15h a t es'-'C with a male- specific JZP labeled (specifi c activity 4xlcP cpm/jjg) nick-translated phage lambda clone H34. Exposure time 3 days. Panel 2: lQpg of Hind 111 digested SacrharomvceB cerevlsiae genomic DNA transferred and hybridized for "15h a t 65 ^ with P- labele d (specific activity 2x10° cpm/pg) nick-translated MAT clon e 2XDX. Exposed for 20h. by hybridizing duplicate Alul blot s of mouse DNA using either Denhard's solution , as conventionally employed, or heparin as described here, under othervise uniform conditions. I t was found that heparin gave a comparatively cleaner background and a subjectively stronger signal, although this may not be apparent from the illustration (Fig.3). As might be expected, hybridizatio n in the presence of Denhard's solution and added carrie r DNA combined with heparin did not materially affect the result. To tes t whether the heparin method works equally well using various commercially available filter membranes, Alul restricted male and female mouse DNA was electrophoretically transferred under identical conditions to thre e differen t examples of filte r membrane (Schleicher & Schull, Gene Screen and Pall Biodyne). These were hybridized in the same annealing reactio n with a single-stranded probe (referred to as 2(8)) which is quantitativel y male-specific (13). The resul t i s shown in Fig.4 in which 5632 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research cf 9 cr 9 using two different methods: (a).Hybridized in the presence of heparin and dextran sulphate as described in the text. (b). Prenybridized overnight in lOxDenhardt solution (lOx is 0.2% each of bovine serum albumin, Ficoll, and polyvinylpyrolidone), 0.1% sodium pyrophosphate, 0.2% SDS, 4xSET, 10% dextran sulphate and 100 jug/ml sheared denatured E.coli DNA). The hybridization mix contained 4xDenhard's solution together with the probe [as in (a)l but was otherwise identical to the prehybridization solution. Hybridization was for 12h at 65°C and washing as described in the text. panels 1 and 2 refer to Schleicher & Schull membrane, respectively with, and without, DNA depurination prior to transfer. Panels 3 and 4 refer, respectively, to Gene Screen and Pall Biodyne; the DNA being depurinated prior to transfer. There was no non-specific background on any of the three filte r types. I t is apparent that depurination, as expected, increased the transfe r efficiency of high molecular weight fragments. However, surprisingly, there was a very striking difference in the hybridization signal strength obtained with the filters from different manufacturers. This 6633 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research o cf <f o <f o C Figure 4. (kig of Alu 1 digested male and female mouse genoraic DNA fractionate d on a 1% agarose gel which was then cut into strips and transferred under identical conditions onto the following filter membranes for hybridization. Panel 1: Schleicher & Schull filte r membrane afte r HC1 depurination of the DNA; Panel 2: Schleicher & Schull filter membrane without depurination; Panel 3: Gene Screen hybridization transfer membrane after HC1 depurination; Panel 4:Pall Biodyne transfer membrane after HC1 depurination. All filter s were hybridized collectively with-single-stranded 3£V labeled (specific activity JxlcP cpm/ug) 2(8) probe, lxlCr cpm/ml. The hybridization mixture contained 4xSET, (11% sodium pyrophosphate, Cl2% SDS, 5Qjq/ml heparin and 1x10° cpm/ml of the probe. No dextra n sulphate was used. Hybridization was a t 65 ^ for 18h. Filters were exposed for 24h. Note absence of background but differences in hybridization signal strength between the filter samples which is highest on Schleicher & Schull. Retention of high molecular weight DNA fragments is relatively poor on Gene Screen and poorest on Pall Biodyne transfer membrane. was not due to the use of heparin but appears to reflect differences in the efficiency with which high molecular weight DNA was retained by the different membranes under our conditions. The experiments were then repeated using the transfe r method of Southern (21) with the same result , thus ruling out the possibilit y that such differential retention was due to the electroblotting procedure. However, electroblotting was more efficien t than the Southern 5634 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research procedure. These experiments were not designed to investigate this point but thi s result was highly repeatable between different samples of these filters. The sample which performed least well (Pall Biodyne) does not show the sex differenc e which i s obvious on the most efficient sample (Schleicher & Schull) after the shorter exposure time. However, equivalent autoradiographs were obtained when the membranes having the poorer performance were exposed for 5 times as long, as shown in Fig.4. Although each manufacturer specifies differen t conditions for efficien t transfer of DNA, which our experiments were not designed to compare, our subsequent experience is that comparable differences in efficiency between these filter samples were stil l evident when the manufacturer's recommendations are observed. Gene Screen Plus™ however, was subsequentl y found to be markedly superior to Gene Screen in terms of the efficiency of DNA transfe r and comparable in this respect with the performance of Schleicher & Schull membranes. According to the manufacturer's recommendations Gene Screen Plus™ does not require Denhardt's treatmen t and we have not ye t fully investigated it s use in the heparin method. In situ hyhridization. I n fiitu annealing of sperm smears with nick translated uncloned Bkm, with single-stranded 2(7) or 2(8) probes (13) or with control probes of pBR322 or phage lambda DNA, revealed consistent and intens e non-specific binding to sperm heads. Other cell types present in the smears did not show such binding (Fig.5a). Deproteinization of the smears (see materials & methods) was ineffective in reducing this non-specific binding. However, i t was abolished by the inclusion of heparin in the hybridization procedure. The optimal concentration s of heparin as established by blot hybridization were employed. Thus, the denatured sperm preparations were pretreated in 5Qjg/ml of heparin and eithe r 50pg/ml or 5OQug/ral of heparin was included in the hybridization mix depending,respectively, on whether or not dextran sulphate wa6 used (Fig.5b,c,d). In these preparations hybridization to specific regions of a proportion of the sperm nuclei can be seen against a background of hybridization (arrows). This probably reflects the fact that Bkm sequences ar e preferentially localised and concentrated in the proximal sex- determining region of the mouse Y chromosome (6,13,14,15,16,,17,). Thus, by thi s approach i t may prove possible cytologically to differentiate Y- and X- bearing mouse sperm. In the preparations shown in Fig.5 some sperms have remained unlabeled (Fig.5C). The source of this effect is unknown but i t might be due to variability in the thermal denaturation of DNA in individual sperm. All three Bkm-related probes described in thi s work gave similar results . DISCUSSION The discovery of Nygaard & Hal l (18) tha t nitrocellulos e filter s strongly adsorbe d single-stranded DNA, along with any hybridized RNA, led to the filte r hybridization method in which DNA was irreversibl y fixed to such 5635 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research Figure 5. Mouse sperm smears hybridized in fiitu with ^H-labeled, nick- translated uncloned Bkm probe as described in the Methods section, (a) Note non-specific binding of probe to the entire sperm heads but not to other cell types in the absence of heparin. 10 days exposure, (b) as in (a) but with heparin in the hybridization mix. Note hybridization now restricted to sperm head nucleus some of which show high concentrations of grains in a localized region of the nucleus (arrow) which may indicate the presence of the Y chromosome.(c) as in (b) but hybridized with single-stranded probe 2(8). arrows: sperms with concentrations of hybridization, (d) as in C showing two sperms side by side differing in hybridization pattern. 6 weeks exposure. membranes before hybridization by drying at moderate temperatures (19). Denhardt (1) found that the non-specific adsorbtion of single-stranded DHA was prevented, without blocking specific hybridization, by pre-incubating the filter s in 0.02% each of Ficoll, polyvinylpyrrolidone and bovine albumin in 3xSSC He pointed out, however, that concentrations of bovine albumin above 0.05% reduced the specific binding of DNA. Denhardt's solution nevertheless has been utilised for all kinds of blot hybridizations, often at concentrations of 10 times that of the pre-incubation mix. Denhardt's solution , does not, however, consistently produce background-free hybridization and, reflecting efforts to overcome this problem, there have been many modifications to the original procedure. It is evident from this alone that the precise way in which non-specific DNA binding to nitrocellulos e is reduced iB unknown. In our experience clean, background- free, blot hybridization can sometimes be obtained in the absence of 5636 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research ingredient s in the annealing solution which are customarily included to contro l background. This fact suggests that the problem of non-specific autoradiographi c background is not inherent in filte r hybridization but probably originates in various impurities which increase the affinity of the probe for the membrane substrate. The protocol described in the present paper uses heparin to reduce non- specific adsorbtion of nucleic acid probes to filters. The heparin effect may be based on the fact that it exhibits an essentially stochiometric binding affinit y for DNA-binding proteins. This forms the basis of the use of heparin in the general heparin-agarose column chromatography method for purifying restriction endonucleases (3). Heparin wil l compete with DNA for protein binding and will inhibit the specific enzymatic functions of some DNA-binding proteins if added to a reaction before, but not after the DNA (4, 5). In thi s respect, therefore, heparin can be regarded as an analogue of DNA. The role of this heparin effect in controlling hybridization background probably lies in the fact that, whereas pure double-stranded DNA wil l not bind to nitrocellulose , the complex of DNA wit h certain proteins binds avidly to nitrocellulose. This effect is quite specific and has been used to investigate the precise nature of complex formation between DNA and site-specifi c endonucleases (5). Moreover, the more impure the preparation of restriction enzyme, the more DNA i s bound (20), implying that the effect i s not limited to restriction enzymes. The nitrocellulos e binding is abolished by heparin if added before the enzyme is permitted to complex with th e DNA. Under many conditions of hybridization there will be some renaturatio n to yiel d duplex radiolabelled DNA prob e molecules. Heparin would be expected to form a complex with any substances which might alter the nitrocellulose binding properties of this proportion of the renatured DNA probe. Also, it cannot be ruled out that some impurities might influence the nature of binding between single-stranded nucleic acids and nitrocellulose under annealing conditions, so producing non-specific background effects. If the heparin effect does operate in respect of single-stranded as well as double-stranded DNA, the order in which the probe is added to the reaction relativ e to the heparin may be critical , but we have not investigated this point. In our procedure, heparin is present in the annealing mix before the probe i s added. A furthe r property of heparin, by analogy with single- stranded DNA, might include a binding capacity for nitrocellulose. If so it would also occupy sites on the filter membrane which normally adsorb DNS and it s complexes but we have not investigated this possibility. ACKNOWLEDGEMENTS We thank Ms.Deidre Hay for excellent technical assistance. This work was supported by the Medical Research Council. 5637 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research REFERENCES 1 . Denhardt,D.T.(1966) Biochem.Biophys.Res.Commun. 23,641-646. 2 . Maitland,N.J., Kinross,J.H., Busuttil,A., Ludgate,S.M., Smart,G.E., & Jones,K.W. (1981) J.Gen.Virol. 55,123-137. 3 . Bickle,T.A.,Pirotta,V., & Imber,R. (1977) Nucleic Acids Res.4, 2561-2572. 4 . Zilllg,W., Zechel,K., Rabussay,D., Schachner,H.,Sethi,V.S., Palm,P., Heil,A.,& Seifert,W. (1970) Cold Spring Harb.Syrap.Quant.Biol. 35, 47-58. 5 . Yuan,R., & Bickle,T.A.,Ebbers,W. , & Brack, C (1975) Nature 256,556-560. 6 . Singh,L. & Jones.K.W. (1982) Cell 28, 205-216. 7 . Bittner,M., Kupferer,P., & Morris,C.F . (1980) Anal.Biochem. 102,459-471. 8 . Wahl,G.M., Stern,M., & Stark,G.R . (1979) Proc. Natl . Acad. Sci . U.S. 76 , 3683-3687. 9 . Maniatis,T., Sim,G.K., Efstratiadis,A and Kafatos,F.C. (1976) Cell 8, 163-182. 10 . Hu,N., & Messing,J. (1982) Gene 17, 271-277. 11 . Jones,K.W. (1973) In New Technique s in Biophysic s and Cel l Biology 1. (R.H.Pain & B.J.Smith Eds.) London: John Wiley & Sons, pp29-66. 12 . Singh,L. Purdom,I.F., & Jones,K.W. (1977) Chromosoma 60, 377-389. 13 . Singh,L., Phillips,C , & Jones , K.W. (1984) Cell 36, 111-120. 14 . Singh,L., Purdom,I.F., & Jones,K.W. (1981) Cold Spring Harb. Symp. Quant . Biol. 45,805-813. 15 . Jones,K.W. & Singh,L.(1981 ) In Genome Evolution. G.Dover & R.Flavell Eds. London: Academic Press. ppl35-154. 16 . Jones,K.W. & Singh,L . (1981) Hum.Genetu58,46-53. 17 . Jones,K.W., Singh,L., & Phillips,C . (1983) Proc. Vth John Innes Symposium. Genetic Rearrangement. Eds. K.F.Chater, CAXullis, EU^Hopwood, AAW.BJohnston & H.W.Woolhouse. Croom Helm London & Canberra.pp265-287. 18 . Nygaard,A.P. & Hall,B.D . (1963) Biochem.Biophy6Jtes.Coramun 12,98-104. 19. Gillespie,a, & Spiegelman,S . (1965) J.Mol.Biol. 12,829-842. 20. Yuan,R., & Meselson,M. (1970) Proc. Natl. Acad. Sci. U.S. 65,357-362. 21 . Southern,E.M. (1975) J.Mol.Biol.98, 503-517. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

The use of heparin as a simple cost-effective means of controlling background In nudeic acid hybridization procedures

Singh,, L.; Jones,, K.W.
Nucleic Acids Research , Volume 12 (14) – Jul 25, 1984
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Oxford University Press
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Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Volume 12 Number 14 1984 Nucleic Acid s Research The use of faeparln as a simple cost-effective means of controlligg background in nucleic add hybridization procedures L.Singh and K.W.Jones Institute of Animal Genetics, University of Edinburgh, Edinburgh EH9 3JN, UK Received 16 May 1984; Revised and Accepted 3 July 1984 ABSTR/CT The use of heparin as a simple, effective and cheap substitute for conventional methods of controlling background in hybridization procedures is described and illustrated with reference to the use of DNA probes in filter and In s_itu hybridization. Possible specific mechanisms for this heparin effect are discussed. INTRODUCTION Refractory background radioactivity in nucleic acid filter hybridization varies in severity with different types of probes, annealing conditions and filte r membranes. Background problems are also encountered in i n fiiili hybridization where they are possibly even more intractable because of the unavoidable complexity in the substrate and the fact that vigorous non- specific methods of control, such as Denhardt's (1) solution cannot be used without damage to the cytological structure. The possibility of a relatively specific means of background control in in situ hybridization was suggested by Maitland et al (2). These workers described non-hybridization binding of labeled DMA probes which was specific to certain cell types. Such binding was substantially reduced by the inclusion in the annealing reaction of the polyanionic glycan, heparin but the optimal conditions for this effect were not determined. Heparin has a substrate binding affinity similar to that of DNA, especially with respect to certain proteins (3). Furthermore, it can abolish the binding of proteins which increase the affinity of DNA for nitrocellulose membranes by specificall y combining with them (4,5). Given this well-characterised property, it is surprising that the possible use of heparin controlling non-specific substrate binding of DNA in both filter and in in situ hybridization apparently has not been explored previously. Accordingly we have examined this aspect and have shown that the inclusion of heparin in the hybridization annealing solution alone can dispense entirely with the use of Denhardt's solution and carrier DNA- This modification result s in the consistent and complete abolition of background in both filte r and i n situ hybridization under a variety of different conditions. Moreover the modified hybridization protocol is much simpler, faster and © IRL Press Limited, Oxford, England. 5627 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research alBO considerably cheaper than existin g methods. MATERIALS AND METHODS DNA extraction . DNA from liver s of male and female mice was isolated by homogenizing the tissu e in TES buffer (3OraH Tris , 5mM EDTA, 50mM NaCl, pH.8.O), lysing the cell s with sarkosyl NL3O (Geigy) and by isopycnic centrifugation in CsCl- ethidium bromide gradients as previously described (6). DNA restrictio n and gel electrophoresis. Restrictio n enzymes and DNA polymerase were obtained from Boehringer Mannheim, New England Biolabs and Bethesda Research Laboratories. DNA was digested by incubating at a ratio of l^ig DNA t o 2 units of enzyme for 4-5h in the buffers and conditions specified by the manufacturer. 0.6 to 1% agarose gel s were run in a vertica l floating gel apparatus at 30V for ll-12h in a buffer containing 15raM Tris, 18mM NaH PO and 0.5mM EDTA, pH 7.8. 2 4 R1p<-+rophoretic transfer of nucleic acids. Electrophoretic transfer of gel-fractionated DNA t o nitrocellulose sheets (7) was carrie d out using a Hoeffer transfer unit after depurination (8). DNA restrictio n fragments were transferred from 2.5 mm thick agarose slab gels by th e following procedure: the ge l was gently shaken for 15 min in 250 ml of O.25M HC1, washed gently with distilled water to remove the residual acid, denatured in two changes 15 min each of 250 ml 0.2M NaOH and 0.6H NaCl and neutralize d in 3 changes each of 500 ml of transfer buffer (TB; O.O25H Na2HPO j/NaH2PO4, pH 6.5). The transfer was carried out in TB a t room temperatur e for lh at 27V keeping the unit cool with circulating cold tap water . The efficiency of transfer was assessed comparatively using the following membranes: Schleicher and Schull membrane filters RefJto. 402097, pore siz e 0.1 um; Gene Screen hybridizatio n transfer membrane Cat.No. NEF- 972, Lot No. 354 GS21; Pall Biodyne transfer membranes P/N BNNG3R, rating 1.2 micron, Lot No. 172340. After transfer, the membranes were carefully washed with TB t o remove residual agarose, dried at room temperature and baked at 8O°C for 2-3 h. Blot hybridization. The following stock solutions were prepared: 20x SET (0.5M NaCl, 0.03H Tris, 2mM EDTA, pH 7.4); 5O% W/V sodium dextran sulphate aqueous solution, stored a t 4-5°C; 50 mg/ml heparin sodium sal t grade II from porcine intestinal mucosa (Sigma,H-7OO5) in 4xSET, stored at 4-5°C for several months; 20% W/V sodium pyrophosphate; 2O% W/V sodium dodecyl sulphate (SDS). i) Hybridization without dpxtran sulphate: The filte r was firs t soaked in 4xSET and placed in a plasti c bag with 10ml of hybridization solution per 10xl4cm membrane. Hybridization salt solution contained 4xSET, 0.1% sodium pyrophosphate solution, 0^% 33S, 50 pq/ml heparin. To avoid the formation of ai r bubbles the solution was preheated to 65°C before use. After heat sealing, the bag was placed in a shaking distilled water bath and incubated with vigorous shaking at 65°C for 2-3h. During thi s time the hybridization 5628 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research bag was kept submerged by another water-filled plastic bag. The denatured, or single-strande d probe was then added to the bag without introducing air bubbles. The bag was then resealed and the solution throughly mixed. The bag was returned to the water bath at 65°t and incubation with vigorous shaking continued for 4-15h depending on the desired C T. For convenience, incubation was normally continued overnight. Ai l Hybridization in the presence of dextran Bulphai-e. The procedure is basically the same as described above except that the solution contained 10% dextran sulphate and lOx the concentration of heparin (500jig/ml). iii ) Washing membrane filters . After either hybridization procedure, the filte r was removed from the hybridization bag, briefl y dipped in 2xSET a t room temperature, re-sealed in a fresh plastic bag with 40O-50Oml of prewarraed wash buffer (2xSET,(X2% SDS) and vigorously agitated for 30min at 65°C The wash buffer was replaced with prewarmed fresh buffer and the step repeated. In the third, and final, washing the wash buffer was replaced with prewarmed buffer adjusted in SET concentration so as to achieve the desired stringency , in the work described here lxSET, 0.2% SDS, and agitated as before. Finally, the membrane was rinsed in 2xSET at room temperature, air drie d and autoradiographed. Filter s can be re-used after removal of the hybridized probe by washing in 0.1% wash buffer (lx wash buffer contains 50mM Tris-HCl, pH 8.0, 2raM EDTA, 0.5% sodium pyrophosphate) for 2h at 65°C Double-stranded DNA was labeled by nick-translation according to the method of Maniatis et al.(9), with minor modifications, using 32P-dCTP (sj«ct. 410 Ci/mM,) or 32P-dATP (sp^ct. 3000 Ci/raM, Padicchemical Centre, Amersham). The reactio n was stopped by adding 5>il of 0.5H EDTA (in the tota l incubation volume of 20ul). 10-20ug of E.coli carrier DNA was then added and the DNA precipitate d with 60>il of absolute ethanol (2 x volume) at -20°C for 2h or -70°C for 30min. Unincorporated 32P dCTP was removed by 3-4 washes in absolut e ethanol in a microcentrifuge. Finally, the precipitate was lyophilised , dissolved in 0.2ml of O.lxSET, denatured for 20min in boiling water , cooled rapidly on ice and used immediately or stored at -20°C. Occasionally, we have used a nick-translated probe without first removing the unincorporated 32P dCTP without experiencing any autoradiographic background problens. Preparation of single-stranded DNA probes. Single stranded DNA probes were prepared according to the method of Hu and Messing (10). A 16 base primer CACAATTCXaCACAAC complementary to the region 5" to the multiple cloning sites of M13rap7, M13mp6 and M13mp9 (New England Biolabs.) was used to initiate limited downstream synthesis of part of the complementary strand of the M13 sequence, leaving the cloned insert single- stranded- This partially single stranded probe labeled with 32P dATP (sp^ict. 3000 Ci/mM) had a specific activity of 3X1O8 cpm/ug. CvtQ.1onjc.a.J preparations. Microscope slide preparations were made from highly concentrated drops of mouse spermatozoa recovered by squeezing the dissected vas deferens in 0.9% 5629 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research NaCl solution. In some samples, the sperm were treated with hypotonic O.O75M KC1 solution for 8 min at 37°C Shears were fixed in 3:1 methanol-acetic acid for 30 min, air dried and stored at 4°C In situ hybr^d.jzation. To make the sperm nuclei accessible to the probe, the slides were incubated i n 50mH dithiothreito l (DTD in 0.1M Tris-HCl buffer, pH 8.0 at room temperatur e for 20 min., rinsed in 2xSSC (SSC i s 0.15M NaCl, 0.015M sodium citrate , pH 7.0), dehydrated through an (50%-100%) alcohol series and air dried. To remove histone and non-histone proteins, the slides were immersed i n a solution containing dextran sulphate (2rag/ml, Pharmacia), heparin (C2mg/ral, Sigma), lOraH Tris (pH 9.0), lOmM H7TA and OJ.% Nonidet P-40 at 5°C for 45 min, rinsed through two 15 min changes of 2xSSC , dehydrated through an ethanol series ,air dried and heat denatured (6). Air dried slide s were rinse d briefl y in heparin solution (50ug/ml in 1^0) and ai r dried without further rinsing. The nick translated DNA probe was denatured, lyophilised and diluted to the desired volume and salt concentration immediately before use. The hybridization mix contained nick-translated DNA (uncloned Bkm; Bkm clone 2(8), or lambda phage DNA), 1.7 ug/ml, sp.act. between 2xlO7 and 4xlO7 cpm/ug; 3xSSC; 10% dextran sulphate and 500 ug/ml heparin. No carrie r DNA was used. Where dextran sulphate was not used the concentration of heparin was reduced to 50 ug/ml. For in situ hybridization the general procedure of Jones (11) was followed with minor modifications. 6 ul of hybridization mix was used on each slide and annealing was carried out at the Tj^ . (60°C) for 4 hr. The hybridized slides were washed in 2xSSC at 60°C for In., washed overnight in 2xSSC a t 4°C, passed through an ethanol series and ai r dried. Hybridized slide s were dipped in Ilford K2 nuclear emulsion, exposed for 1 to 10 weeks, developed in Kodak Dl9b developer, fixed in Ilford Hypara, stained in Giemsa and photographed as described previously (12). RESITr.TS- Blot hybridization. Heparin concentrations over the range of 5 t o 10OOpg/ml were teste d in order to establish the minimum concentration effective in abolishing background in the absence of other measures. As might be expected, the higher the effective concentration and the specific activity of the probe the more hepari n needed to effectively supress background.At the probe specific activitie s achieved (5xlO7-3xlO^ cpm/ug),and with added probe of from 2xlO5-2xlO6 cpm/ml,it was found necessary to add 5O-200 jjg/ml of heparin in the absence of dextran sulphate. This was required to be increased to 500-700 .ug/ml of heparin in the presence of dextran sulphate. The tenfold higher concentration presumably is necessary to offset the concentrating effect of dextran sulphate on the probe. To obtain complete control of background it was found effective to include heparin only in the hybridization mix. Hybridization in dextran sulphate of the single-stranded cloned Bkm probe 2(8) (13) to an Alul digested mouse DNA blo t is shown in Fig 1. with and 5630 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research o CT ^ C Figure 1. (yjl of male and female mouse genomic DNA digeste d with Alul, fractionated on 1% agarose gel, transferred onto Schleicher & Schull membrane filter s and hybridized with, the -"P single-stranded probe 2(8) 6xlCP cpra/ml (specifi c activity 3.2x10° cpm/ug) for 12h at 65°C (see Experimental Procedures) exposed for 15h without pre-sensitizing the film. Note absence of background in panel 1 which was hybridized in the presence of dextran sulphat e and heparin. Panel 2 shows the same sequences hybridized under identical conditions in the absence of heparin. Note patchy background. without 50Qug/ml of heparin (panels 1 and 2 respectively). Heavy patchy background occurs only in the filte r hybridized in the absence of heparin. Hybridization to low copy number sequences in mouse and yeast genomic DNA using the heparin/dextran sulphate method is shown in Fig.2 . Panel 1 shows EcoRl digested male and female mouse DNA hybridized with a probe of a mouse male-specifi c fragment (H34) recovered from a mouse genomal library. The absence of background is evident from the female DNA track which lacks the sequence in question. Panel 2 shows yeas t DNA hybridize d with a cloned MAT sequence probe (2DX) and is , similarly, free from non-specific background. A brie f comparative assessment of the heparin and Denhardt methods was made 5631 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research er o. M34 2DX Figure 2. Hybridization of cloned probes of single copy sequences to genomic DNA i n the presence of heparin and dextran sulphate snowing absence of background.Panel 1: 6>jg of EcoRl digested male and female jnouse DNA transferred and hybridized for 15h a t es'-'C with a male- specific JZP labeled (specifi c activity 4xlcP cpm/jjg) nick-translated phage lambda clone H34. Exposure time 3 days. Panel 2: lQpg of Hind 111 digested SacrharomvceB cerevlsiae genomic DNA transferred and hybridized for "15h a t 65 ^ with P- labele d (specific activity 2x10° cpm/pg) nick-translated MAT clon e 2XDX. Exposed for 20h. by hybridizing duplicate Alul blot s of mouse DNA using either Denhard's solution , as conventionally employed, or heparin as described here, under othervise uniform conditions. I t was found that heparin gave a comparatively cleaner background and a subjectively stronger signal, although this may not be apparent from the illustration (Fig.3). As might be expected, hybridizatio n in the presence of Denhard's solution and added carrie r DNA combined with heparin did not materially affect the result. To tes t whether the heparin method works equally well using various commercially available filter membranes, Alul restricted male and female mouse DNA was electrophoretically transferred under identical conditions to thre e differen t examples of filte r membrane (Schleicher & Schull, Gene Screen and Pall Biodyne). These were hybridized in the same annealing reactio n with a single-stranded probe (referred to as 2(8)) which is quantitativel y male-specific (13). The resul t i s shown in Fig.4 in which 5632 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research cf 9 cr 9 using two different methods: (a).Hybridized in the presence of heparin and dextran sulphate as described in the text. (b). Prenybridized overnight in lOxDenhardt solution (lOx is 0.2% each of bovine serum albumin, Ficoll, and polyvinylpyrolidone), 0.1% sodium pyrophosphate, 0.2% SDS, 4xSET, 10% dextran sulphate and 100 jug/ml sheared denatured E.coli DNA). The hybridization mix contained 4xDenhard's solution together with the probe [as in (a)l but was otherwise identical to the prehybridization solution. Hybridization was for 12h at 65°C and washing as described in the text. panels 1 and 2 refer to Schleicher & Schull membrane, respectively with, and without, DNA depurination prior to transfer. Panels 3 and 4 refer, respectively, to Gene Screen and Pall Biodyne; the DNA being depurinated prior to transfer. There was no non-specific background on any of the three filte r types. I t is apparent that depurination, as expected, increased the transfe r efficiency of high molecular weight fragments. However, surprisingly, there was a very striking difference in the hybridization signal strength obtained with the filters from different manufacturers. This 6633 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research o cf <f o <f o C Figure 4. (kig of Alu 1 digested male and female mouse genoraic DNA fractionate d on a 1% agarose gel which was then cut into strips and transferred under identical conditions onto the following filter membranes for hybridization. Panel 1: Schleicher & Schull filte r membrane afte r HC1 depurination of the DNA; Panel 2: Schleicher & Schull filter membrane without depurination; Panel 3: Gene Screen hybridization transfer membrane after HC1 depurination; Panel 4:Pall Biodyne transfer membrane after HC1 depurination. All filter s were hybridized collectively with-single-stranded 3£V labeled (specific activity JxlcP cpm/ug) 2(8) probe, lxlCr cpm/ml. The hybridization mixture contained 4xSET, (11% sodium pyrophosphate, Cl2% SDS, 5Qjq/ml heparin and 1x10° cpm/ml of the probe. No dextra n sulphate was used. Hybridization was a t 65 ^ for 18h. Filters were exposed for 24h. Note absence of background but differences in hybridization signal strength between the filter samples which is highest on Schleicher & Schull. Retention of high molecular weight DNA fragments is relatively poor on Gene Screen and poorest on Pall Biodyne transfer membrane. was not due to the use of heparin but appears to reflect differences in the efficiency with which high molecular weight DNA was retained by the different membranes under our conditions. The experiments were then repeated using the transfe r method of Southern (21) with the same result , thus ruling out the possibilit y that such differential retention was due to the electroblotting procedure. However, electroblotting was more efficien t than the Southern 5634 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research procedure. These experiments were not designed to investigate this point but thi s result was highly repeatable between different samples of these filters. The sample which performed least well (Pall Biodyne) does not show the sex differenc e which i s obvious on the most efficient sample (Schleicher & Schull) after the shorter exposure time. However, equivalent autoradiographs were obtained when the membranes having the poorer performance were exposed for 5 times as long, as shown in Fig.4. Although each manufacturer specifies differen t conditions for efficien t transfer of DNA, which our experiments were not designed to compare, our subsequent experience is that comparable differences in efficiency between these filter samples were stil l evident when the manufacturer's recommendations are observed. Gene Screen Plus™ however, was subsequentl y found to be markedly superior to Gene Screen in terms of the efficiency of DNA transfe r and comparable in this respect with the performance of Schleicher & Schull membranes. According to the manufacturer's recommendations Gene Screen Plus™ does not require Denhardt's treatmen t and we have not ye t fully investigated it s use in the heparin method. In situ hyhridization. I n fiitu annealing of sperm smears with nick translated uncloned Bkm, with single-stranded 2(7) or 2(8) probes (13) or with control probes of pBR322 or phage lambda DNA, revealed consistent and intens e non-specific binding to sperm heads. Other cell types present in the smears did not show such binding (Fig.5a). Deproteinization of the smears (see materials & methods) was ineffective in reducing this non-specific binding. However, i t was abolished by the inclusion of heparin in the hybridization procedure. The optimal concentration s of heparin as established by blot hybridization were employed. Thus, the denatured sperm preparations were pretreated in 5Qjg/ml of heparin and eithe r 50pg/ml or 5OQug/ral of heparin was included in the hybridization mix depending,respectively, on whether or not dextran sulphate wa6 used (Fig.5b,c,d). In these preparations hybridization to specific regions of a proportion of the sperm nuclei can be seen against a background of hybridization (arrows). This probably reflects the fact that Bkm sequences ar e preferentially localised and concentrated in the proximal sex- determining region of the mouse Y chromosome (6,13,14,15,16,,17,). Thus, by thi s approach i t may prove possible cytologically to differentiate Y- and X- bearing mouse sperm. In the preparations shown in Fig.5 some sperms have remained unlabeled (Fig.5C). The source of this effect is unknown but i t might be due to variability in the thermal denaturation of DNA in individual sperm. All three Bkm-related probes described in thi s work gave similar results . DISCUSSION The discovery of Nygaard & Hal l (18) tha t nitrocellulos e filter s strongly adsorbe d single-stranded DNA, along with any hybridized RNA, led to the filte r hybridization method in which DNA was irreversibl y fixed to such 5635 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research Figure 5. Mouse sperm smears hybridized in fiitu with ^H-labeled, nick- translated uncloned Bkm probe as described in the Methods section, (a) Note non-specific binding of probe to the entire sperm heads but not to other cell types in the absence of heparin. 10 days exposure, (b) as in (a) but with heparin in the hybridization mix. Note hybridization now restricted to sperm head nucleus some of which show high concentrations of grains in a localized region of the nucleus (arrow) which may indicate the presence of the Y chromosome.(c) as in (b) but hybridized with single-stranded probe 2(8). arrows: sperms with concentrations of hybridization, (d) as in C showing two sperms side by side differing in hybridization pattern. 6 weeks exposure. membranes before hybridization by drying at moderate temperatures (19). Denhardt (1) found that the non-specific adsorbtion of single-stranded DHA was prevented, without blocking specific hybridization, by pre-incubating the filter s in 0.02% each of Ficoll, polyvinylpyrrolidone and bovine albumin in 3xSSC He pointed out, however, that concentrations of bovine albumin above 0.05% reduced the specific binding of DNA. Denhardt's solution nevertheless has been utilised for all kinds of blot hybridizations, often at concentrations of 10 times that of the pre-incubation mix. Denhardt's solution , does not, however, consistently produce background-free hybridization and, reflecting efforts to overcome this problem, there have been many modifications to the original procedure. It is evident from this alone that the precise way in which non-specific DNA binding to nitrocellulos e is reduced iB unknown. In our experience clean, background- free, blot hybridization can sometimes be obtained in the absence of 5636 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research ingredient s in the annealing solution which are customarily included to contro l background. This fact suggests that the problem of non-specific autoradiographi c background is not inherent in filte r hybridization but probably originates in various impurities which increase the affinity of the probe for the membrane substrate. The protocol described in the present paper uses heparin to reduce non- specific adsorbtion of nucleic acid probes to filters. The heparin effect may be based on the fact that it exhibits an essentially stochiometric binding affinit y for DNA-binding proteins. This forms the basis of the use of heparin in the general heparin-agarose column chromatography method for purifying restriction endonucleases (3). Heparin wil l compete with DNA for protein binding and will inhibit the specific enzymatic functions of some DNA-binding proteins if added to a reaction before, but not after the DNA (4, 5). In thi s respect, therefore, heparin can be regarded as an analogue of DNA. The role of this heparin effect in controlling hybridization background probably lies in the fact that, whereas pure double-stranded DNA wil l not bind to nitrocellulose , the complex of DNA wit h certain proteins binds avidly to nitrocellulose. This effect is quite specific and has been used to investigate the precise nature of complex formation between DNA and site-specifi c endonucleases (5). Moreover, the more impure the preparation of restriction enzyme, the more DNA i s bound (20), implying that the effect i s not limited to restriction enzymes. The nitrocellulos e binding is abolished by heparin if added before the enzyme is permitted to complex with th e DNA. Under many conditions of hybridization there will be some renaturatio n to yiel d duplex radiolabelled DNA prob e molecules. Heparin would be expected to form a complex with any substances which might alter the nitrocellulose binding properties of this proportion of the renatured DNA probe. Also, it cannot be ruled out that some impurities might influence the nature of binding between single-stranded nucleic acids and nitrocellulose under annealing conditions, so producing non-specific background effects. If the heparin effect does operate in respect of single-stranded as well as double-stranded DNA, the order in which the probe is added to the reaction relativ e to the heparin may be critical , but we have not investigated this point. In our procedure, heparin is present in the annealing mix before the probe i s added. A furthe r property of heparin, by analogy with single- stranded DNA, might include a binding capacity for nitrocellulose. If so it would also occupy sites on the filter membrane which normally adsorb DNS and it s complexes but we have not investigated this possibility. ACKNOWLEDGEMENTS We thank Ms.Deidre Hay for excellent technical assistance. This work was supported by the Medical Research Council. 5637 Downloaded from https://academic.oup.com/nar/article/12/14/5627/1203737 by DeepDyve user on 20 August 2020 Nucleic Acids Research REFERENCES 1 . Denhardt,D.T.(1966) Biochem.Biophys.Res.Commun. 23,641-646. 2 . Maitland,N.J., Kinross,J.H., Busuttil,A., Ludgate,S.M., Smart,G.E., & Jones,K.W. (1981) J.Gen.Virol. 55,123-137. 3 . Bickle,T.A.,Pirotta,V., & Imber,R. (1977) Nucleic Acids Res.4, 2561-2572. 4 . Zilllg,W., Zechel,K., Rabussay,D., Schachner,H.,Sethi,V.S., Palm,P., Heil,A.,& Seifert,W. (1970) Cold Spring Harb.Syrap.Quant.Biol. 35, 47-58. 5 . Yuan,R., & Bickle,T.A.,Ebbers,W. , & Brack, C (1975) Nature 256,556-560. 6 . Singh,L. & Jones.K.W. (1982) Cell 28, 205-216. 7 . Bittner,M., Kupferer,P., & Morris,C.F . (1980) Anal.Biochem. 102,459-471. 8 . Wahl,G.M., Stern,M., & Stark,G.R . (1979) Proc. Natl . Acad. Sci . U.S. 76 , 3683-3687. 9 . Maniatis,T., Sim,G.K., Efstratiadis,A and Kafatos,F.C. (1976) Cell 8, 163-182. 10 . Hu,N., & Messing,J. (1982) Gene 17, 271-277. 11 . Jones,K.W. (1973) In New Technique s in Biophysic s and Cel l Biology 1. (R.H.Pain & B.J.Smith Eds.) London: John Wiley & Sons, pp29-66. 12 . Singh,L. Purdom,I.F., & Jones,K.W. (1977) Chromosoma 60, 377-389. 13 . Singh,L., Phillips,C , & Jones , K.W. (1984) Cell 36, 111-120. 14 . Singh,L., Purdom,I.F., & Jones,K.W. (1981) Cold Spring Harb. Symp. Quant . Biol. 45,805-813. 15 . Jones,K.W. & Singh,L.(1981 ) In Genome Evolution. G.Dover & R.Flavell Eds. London: Academic Press. ppl35-154. 16 . Jones,K.W. & Singh,L . (1981) Hum.Genetu58,46-53. 17 . Jones,K.W., Singh,L., & Phillips,C . (1983) Proc. Vth John Innes Symposium. Genetic Rearrangement. Eds. K.F.Chater, CAXullis, EU^Hopwood, AAW.BJohnston & H.W.Woolhouse. Croom Helm London & Canberra.pp265-287. 18 . Nygaard,A.P. & Hall,B.D . (1963) Biochem.Biophy6Jtes.Coramun 12,98-104. 19. Gillespie,a, & Spiegelman,S . (1965) J.Mol.Biol. 12,829-842. 20. Yuan,R., & Meselson,M. (1970) Proc. Natl. Acad. Sci. U.S. 65,357-362. 21 . Southern,E.M. (1975) J.Mol.Biol.98, 503-517.

Journal

Nucleic Acids Research – Oxford University Press

Published: Jul 25, 1984

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