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Subtractive hybridization of biotinylated DNA in phenol emulsion
J. Biochem. Biophys. Methods 50 Ž2001. 43–52 www.elsevier.comrlocaterjbbm
Subtractive hybridization of biotinylated DNA in phenol emulsion Alexandr G. Laman, Sergei G. Kurjukov, Elena V. Bulgakova, Nadezhda N. Anikeeva, Feodor A. Brovko ) Branch of Shemyakin and OÕchinnikoÕ Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russian Federation Received 2 December 1999; received in revised form 3 April 2001; accepted 17 May 2001
Abstract A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique ŽPERT. has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5–1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript ŽIL-2. in three cycles of subtractive hybridization. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Subtractive hybridization; Biotinylated DNA; Differential cDNA libraries; Driver DNA; Tracer DNA; PERT
1. Introduction The methods for creating cDNA libraries on the basis of subtractive hybridization techniques have now become very popular w1–5x. Differential cDNA libraries are a very useful instrument for revealing and studying the genes with the regulated expression. The completeness of DNA hybridization, the hybridization time, and the degree of AbbreÕiations: Bio, biotin; Bio-DNA, biotinylated DNA; ExoIII, exonuclease III; IL-2, interleukin-2; PERT, phenol emulsion reassociation technique; PHA, phytohemagglutinin; StrAv, streptavidin ) Corresponding author. Tel.: q7-095-9252342, q7-0967-730853; fax: q7-0967-790527. E-mail address: [email protected] ŽF.A. Brovko.. 0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 1 7 3 - 7
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removal of homologous sequences from the reaction mixture are of critical importance for the process of subtractive hybridization. Thus, any improvements in hybridization techniques are of great importance for research and diagnostic purposes. Due to the high affinity of streptavidin to biotin, biotinylated DNA hybrids can be quickly removed from the reaction mixture w6x. The stability of streptavidin–biotin complexes w7x enables hybridization in a wide pH range Ž4 to 9. and in the presence of organic solvents. The above factors, as well as the simplicity of the DNA biotinylation procedure, make this approach attractive as applied to the method of subtractive hybridization w8,9x. Phenol emulsion reassociation technique ŽPERT. proposed by Kohne et al. w10x is a quick and convenient method of DNA hybridization in water solutions in wide concentration ranges Ž6 = 10y6 to 6400 mkgrml. that significantly increases the rate of DNA reassociation Ž10–1000 times. as compared with hybridization in a single-phase water system. PERT does not require high DNA concentrations ŽDNA solutions of below 4 mkgrml can be used. and significantly reduces the loss of rare sequences. This is particularly vital for rare transcripts, when it is necessary to obtain an enriched cDNA fraction. Nevertheless, hybridization in phenol emulsion was not used in case of biotinylated DNA due to the formation of water-insoluble aggregates of hybrid DNA w5x. We have revealed that the degree and character of DNA biotinylation influence the formation of Bio-DNA aggregates. This study was aimed to optimize the conditions of insertion of biotin residues into DNA and hybridization of the biotinylated DNA as applied to the method of subtractive hybridization in phenol emulsion. The conditions were selected for the enrichment of rare or developmental phase-specific transcripts in a real biological system to estimate the change in the level of gene expression. The method was approved on a model system of enrichment with rare mRNA sequences Žinduced mRNA of interleukin-2..
2. Materials and methods 2.1. Methods of DNA biotinylation 2.1.1. Photobiotinylation For DNA biotinylation, the plasmid pBluescript KS was digested with EcoRI and labelled with wP32x on 5X cohesive ends. A 2-mkg of linearized and denatured plasmid was biotinylated by photoactivated biotin ŽVector laboratories. according to manufacturer’s instruction. 2.1.2. Biotinylation by filling the ends A 2-mkg of the plasmid pBluescript KS linearized by EcoRI was biotinylated at 37 8C for 10 min in 50 mkl of the buffer for Klenow Fragment in the presence of 100 mkM dATP, dGTP, dCTP, bio-4-dUTP, and 5 U of Klenow Fragment Ž10 mM Tris–HCl, pH 8.0, 10 mM MgCl 2 .. To determine the degree of biotinylation, the reaction was performed in the presence of wa-32Px dATP Ž0.3 mkM..
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2.1.3. Biotinylation after partial DNA hydrolysis with ExoIII A 2-mkg of the linearized plasmid was hydrolyzed in 30 mkl of the buffer for Klenow Fragment using 175 U of ExoIII. The time of hydrolysis was chosen to hydrolyze 20–100 nucleotides. The hydrolyzed plasmid was biotinylated using bio-4dUTP by the method described above. Biotinylated DNA was purified from free nucleotides by passing the sample through a minicolumn with Sephadex G25F. 2.2. Hybridization of the biotinylated DNA to the completely homological DNA in phenol emulsion A 200-ng of the biotinylated plasmid pBluescript KS DNA was dissolved in 45 mkl of TE buffer, and then mixed with 200 ng of 5X-w32Px-labelled DNA of the same plasmid. Then the solution was denaturated by boiling for 2 min, supplemented with 45 mkl of 2.5 M lithium perchlorate and 10 mkl of 90% phenol ŽpH 9.0., and hybridized for 1 h at room temperature and with intensive stirring. On hybridization, the water and phenol phases were separated by centrifugation. Nine acetone volumes were added to the water phase, cooled in ice for 5 min, and centrifuged; the pellet was dissolved in 96 mkl of TE buffer Ž10 mM Tris–HCl, pH 7.5, 1 mM EDTA.. The amount of insoluble DNA aggregates formed during hybridization was determined by measuring the radioactivity of equal aliquots Ž0.1 mkl. of the water phase before and after the hybridization. 2.3. Estimation of hybridization degree and completeness of remoÕal of Bio-DNA hybrids and non-biotinylated DNA On hybridization Žthe same as in Section 2.2., DNA was precipitated with ethanol and dissolved in 100 mkl of SSC Ž1 = . buffer; streptavidin was added to a concentration of 2 mkgrml, and in 30 min the complexes of StrAv–Bio-DNA hybrids were removed using phenol:chloroform Ž1:1. extraction. The percentage of remaining and removed DNA was assessed by the radioactivity ratio of water phases before and after the extraction. 2.4. Synthesis and amplification of cDNA 10 5 cells of the line Jurkat were taken to isolate driver RNA. 10 5 cells of the line Jurkat after their stimulation with phytohemaglutinin by the method described by Belyavsky et al. w12x were taken to isolate tracer RNA. The total RNA was isolated from cells by the guanidinium isothiocyanate method w11x. The tracer and driver cDNA were synthesized using a Universal Riboclone cDNA Synthesis System ŽPromega.. Oligonucleotide adapters B49 Ž5X-AATTCGTCGACATCGAT. and B50 Ž5X-ATCGATGTCGACG. were joined to the double-stranded driver cDNA. Adapters B5 Ž5X-ATTCCTGCAGAATTCTCC., B6 Ž5X GGAGAATTCTGCAGG. were joined to the tracer cDNA. The only one round with 30 cycles of amplification with the primers B49 and B5 was performed for the driver and tracer, respectively, in 100 mkl reaction mixture containing 2 mM MgCl2, 0.3 mkM primer, 0.25 mM dNTP, 1 U of Taq polymerase in the following regime: t 8C: 96–1 min, 46–1 min, 72–2 min on a device THC-2 ŽTechne, England..
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2.5. Procedure of subtractiÕe hybridization The first cycle of subtraction: 200 ng of non-biotinylated tracer DNA and 2 mkg of driver DNA biotinylated using bio-4-dUTP were dissolved in 45 mkl of TE buffer, denatured by boiling for 2 min, and supplied with 45 mkl of 2.5 M lithium perchlorate and 10 mkl of 90% phenol ŽpH 9.0.. Hybridization was carried out for 0.5–1 h at room temperature and with intensive stirring. On hybridization, DNA from the water phase was precipitated with 10 volumes of acetone and centrifuged; the pellet was dissolved in 45 mkl of TE buffer. Streptavidin was added to a concentration of 2 mkgrml and stirred during 10 min, then the complexes of biotinylated DNA–streptavidin were removed by extraction with an equal volume of phenol:chloroform Ž1:1, vrv. and ethanol precipitation. The second cycle of subtraction: 1 mkg of biotinylated driver DNA and lithium perchlorate solution were added to the water phase containing tester DNA to a lithium perchlorate concentration of 1.25 M, and phenol to a concentration of 10%. Hybridization was carried out as in the first cycle. The third and fourth cycles of hybridization proceeded in the same way. 2.6. Hybridization in the presence of dextran sulfate Two hundred nanograms of non-biotinylated w32Px-labelled DNA of the linearized plasmid pBluescript KS and 200 ng of DNA of the same plasmid biotinylated using bio-4-dUTP Žas in 2.1. were dissolved in 20 mkl of water, denatured by heating to 95 8C for 5 min, and then hybridized in 200 mkl of 6 = SSC solution containing 10% dextran sulfate for 1 h at 60 8C w11x. After hybridization, DNA was precipitated and dissolved in a 1 = SSC buffer Ž150 mM NaCl, 15 mM Na citrate, pH 7.4.; streptavidin was added to 2 mkgrml, stirred, and after 10-min incubation, the complexes of protein–Bio-DNA were removed by extraction with: Ž1. phenol:chloroform mixture Ž1:1, vrv., Ž2. chloroform, Ž3. ether. The amount of hybridized DNA was assessed by the ratio of water phase radioactivity level before and after the hybridization. 2.7. Blot hybridization After three cycles of subtraction, the tracer cDNA was amplified as described in Section 2.4. A 1-mkg of each cDNA Žtracer, driver, and subtracted tracer cDNA. was separated in 1.5% agarose gel, transferred to a membrane Hybond C Extra ŽAmersham., and hybridized with w32Px-labelled oligonucleotide specific for cDNA IL-2 dŽAGTGCACCTACTTCAAGT.. Electrophoresis in agarose, transfer and hybridization were carried out according to the standard protocol w11x. 2.8. Calculation of the efficiency of subtraction The zones of hybridization filters of equal areas, corresponding to IL-2 cDNA ŽFig. 2., were taken to determine the degree of enrichment; their radioactivity was assessed by scintillation on a counter LS-6800 ŽBeckman.. The radioactivity of the filter site of the corresponding cDNA of the cells not stimulated by PHA was taken as a base line. The
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factor of enrichment Fe was calculated as a ratio of radioactivities of filter zones by the formula Fe s ŽSub y N .rŽStim y N .. In our case, Sub s 16100" 100, Stims 85 " 10, and N s 30 " 10 and enrichment factor s 292.18. 3. Results and discussion The stage of hybridization is the most time-consuming in the gene engineering experiments on the hybridization of nucleic acids. Therefore, the improvement of hybridization methods is an urgent task. We have proposed a method of subtractive hybridization in phenol emulsion using biotinylated DNA. It has the advantages of PERT, significantly shortening the time of hybridization and increasing the rate of homologous DNA reassociation as compared with the standard hybridization in a single-phase water system. This is particularly important in the work with small amounts of available DNA and in DNA hybridization at low concentrations Žless than 40 mkgrml.. Previously, it was considered impossible to use Bio-DNA in PERT w5x. The method we have developed employs both Bio-DNA in PERT and an efficient technique of duplex DNA removal using the streptavidin–biotin system. At first, we studied the formation of water-insoluble Bio-DNA aggregates under the PERT conditions and analyzed the efficiency of removal of Bio-DNA and their heteroduplexes from the reaction mixture after incubation with streptavidin and phenol extraction. The following conditions appeared to be optimal: 10% phenol Žvrv., pH 9.0, and 1.25 M solution of lithium perchlorate as a chaotropic agent. These conditions were used for hybridization of biotinylated DNA. DNA of the linearized plasmid pBluescript KS with biotin residues joined by different methods was chosen as a model system. Bio-DNA was hybridized under the PERT conditions with the same non-biotinylated DNA but labelled with w32Px by the 5X end. The amount of aggregated DNA was assessed by the change in the DNA content in the water phase after separation of the phenol phase. The results of these experiments are given in Table 1. The data show that not only the degree but also the character of biotinylation are the critical parameters affecting the
Table 1 The effect of DNA biotinylation method on the efficiency of subtractive hybridization in phenol emulsion Method of DNA biotinylation
Degree of biotinylation Žbiotin moleculesr DNA molecules.
% of DNA forming water-insoluble aggregates
% of DNA extracted with phenol:chloroform after binding of streptavidin
Photobiotinylation Filling of DNA partially hydrolyzed by ExoIII X Filling of DNA with 5 extending tetranucleotides
ND 8–17
98 82–90
495 495
1–2
-5
495
ND—not determined, n –number of residues Bio per 1 DNA molecule-10 w8x.
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formation of water-insoluble aggregates. The degree of aggregate formation was negligible only at the insertion of one to two biotin molecules into the 3X end of the DNA molecule, and such DNA could be used for hybridization in phenol emulsion. At increasing the biotinylation degree to 8–17 biotin molecules per 3X end of the DNA molecule, the amount of hybrid DNA remaining in the water phase dramatically decreased. The similar process with a still higher percentage of water-insoluble aggregates was also observed with a photobiotinylated DNA, although this method of biotinylation introduces one biotin residue per 300 bp on average w8x. The size of DNA was about 3000 bp; consequently, the quantity of biotin residues per such a molecule was over 10. At the same time, this DNA formed more water-insoluble aggregates than DNA biotinylated by the 3X end of the molecule, which is evidently due to a statistically random character of the photobiotinylation reaction. We assume that the reduced formation of water-insoluble aggregates at hybridization under the PERT conditions for DNA with one to two biotin residues on 3X ends is explained by biotin distribution along the DNA molecule. It is known that at low pH values Ž- 4.2. w13x, the linear DNA molecules pass to the phenol phase under extraction. Cetyltrimethylammonium salt of DNA is extracted by butanol w14x. In both cases, due either to charge neutralization or to the formation of a salt with the aliphatic base, the hydrophobicity increases over the whole DNA molecule, which is responsible for its passing to the organic phase. We have suggested that the formation of aggregates during hybridization under the PERT conditions is associated with the character of biotin distribution over the DNA molecule. The even distribution of biotin may result in the formation of uniformly hydrophobic molecular structures. Thus, DNA acquires the ability to form associates passing to the organic phase, in particular to phenol. It may be assumed that the smaller the region forming the hydrophobic structure, the lower is its affinity to the organic phase and, consequently, the ability of DNA to pass to the latter. We decided to test this assumption with regard to the fact that the DNA hydrophobicity decreases with the pH increase, using three methods of biotinylation: Ž1. photobiotinylation, providing an even distribution of biotin over the DNA molecule; Ž2. partial hydrolysis of the 3X end of DNA in controlled regions followed by the filling of the ends in the presence of a biotinylated analog of nucleoside triphosphate Žbio-4-dUTP.. In this case, 8 to 17 biotinylated nucleotides were incorporated into DNA; Ž3. the filling of EcoRI-digested DNA by Klenow Fragments and biotinylated bio-4-dUTP, with no more than two biotinylated bases incorporated into DNA molecules. Thus, the insertion of one to two biotin residues into the DNA molecule ends appeared to be the best, since this method of biotinylation does not significantly change the physical and chemical properties of DNA under the PERT conditions. The completeness of hybridization, the degree of removal of heteroduplexes and driver DNA, and the time of performance are the most important factors for subtractive hybridization. The completeness of hybridization was analyzed using a w32Px-labelled DNA from the plasmid pBluescript KS hydrolyzed by EcoRI and the same DNA labelled with biotin by filling the cohesive ends. The quantitative analysis of DNA after hybridization showed that about 95% of plasmid DNA is hybridized in 60 min in case of insertion of one to two biotin molecules into the 3X end.
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At present, the hybridization procedure is accelerated by other widely used methods with enhancers, such as dextran sulfate w15x. For comparison, we have chosen a widespread method of DNA solution hybridization in the presence of 10% dextran sulfate w11x. A plasmid DNA biotinylated by the 3X end and a similar DNA labelled with w32Px by the 5X end were taken as a model system. Our data show that only 37% plasmid DNA was reassociated in 60 min of hybridization in the solution with dextran sulfate, i.e., the Bio-DNA hybridization in phenol emulsion is much more efficient than with the simultaneous addition of dextran sulfate. Besides, it is very difficult to purify the subtracted DNA from dextran sulfate admixtures after hybridization to use it in further experiments on cloning and PCRamplification due to the inhibitory properties of dextran sulfate to many enzymes. The results of plasmid DNA hybridization in phenol emulsion using Bio-DNA served as a basis of approaches to the search and enrichment of rare or phase-specific mRNA. The T-cell lane Jurkat stimulated with phytohemaglutinin w16x was used as a model. With such type of stimulation, new mRNA are synthesized, which are extremely few or completely absent in non-stimulated cells w16,17x. The stimulation of T-cells induces the expression of about 100 genes, one of which Žthe gene of interleukin-2. was chosen as a model w16x. cDNA from stimulated cells Žtracer. were labelled with w32Px, and cDNA from non-stimulated cells Ždriver. were biotinylated. The hybridization of amplified cDNA was performed under the PERT conditions with a 10-fold excess of driver DNA in relation to tracer DNA. The analysis of hybridized DNA showed ŽFig. 1. that about 90% are hybridized in 30 min. The second cycle of hybridization reduced the amount of non-hybridized DNA to
Fig. 1. The efficiency of removal of tracer sequences during subtractive hybridization. Axes are the percentage of tracer DNA in solution and the number of subtractive cycles.
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Fig. 2. Blot hybridization of DNA probes Ž1 mkg each. with interleukin-2 specific probe. Lane 1: P32-labelled DNA mass markers 393 and 270 bp Ž30000 and 40000 cpm per band.. Lane 2: Driver DNA. Lane 3: Non-subtracted tracer. Lane 4: Subtracted and amplified tracer.
2.9%. The third and further cycles did not show any reliably detectable changes in the amount of non-hybridized tracer DNA. The data obtained lead to the following conclusion: a great part of frequent sequences is removed during the first cycle of hybridization. Intermediate and rare DNA are removed during the second and further cycles. Thus, one to two cycles of hybridization under the above described conditions are enough for practical work. The decisive criterion of subtractive hybridization is the degree of enrichment with desired sequences. Three cycles of tracer cDNA subtraction with biotinylated driver cDNA were made to determine the degree of enrichment. The degree of enrichment of tracer cDNA was estimated by the ratio of radioactivity of enriched, stimulated and non-stimulated DNA after blot hybridization with a radioactively labelled oligonucleotide probe to cDNA of interleukin-2 and radioautography ŽFig. 2.. As Fig. 2 shows, the zone corresponding to IL-2 cDNA Žlane 1. is actually absent on lane 2 after an overnight exposure. The analysis of radioactivity of equal-squared filter sections corresponding to the subtracted and stimulated DNA showed a 300-fold efficiency of subtraction. The radioactivity of the same filter section of lane 3 of non-stimulated cells was used as a base level of the above calculation. Rare mRNA, e.g., mRNA of transferin receptor, are enriched 20-fold after four cycles of subtraction as shown by Rivolta and Wilcox w18x. In our case, the degree of enrichment about 300-fold was achieved in three cycles of subtraction.
4. A simplified description of the method of Bio-DNA subtractive hybridization in phenol emulsion and its application We have proposed a method of subtractive hybridization using biotinylated DNA and PERT. The above method reduces the time of reassociation from the conventional 24–48 h to 0.5–1 h at the tracer DNA concentration of 4 mkgrml. DNA heterohybrids are removed by a highly efficient method of Bio-DNA extraction as complexes with
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streptavidin. Using different adapter pairs to obtain tracer and driver, DNA permits a selective amplification of the tracer upon the last cycle of hybridization. Besides, the completeness of the driver DNA removal prevents the non-specific amplification and contamination of subtracted DNA. A possibility of high-degree enrichment for rare mRNA sequences was shown in the model system of IL-2 mRNA. The proposed technique does not require high DNA concentrations; the subtractive hybridization can proceed with the volumes commonly used in biochemical practice and at room temperature. The proposed method can be used not only for the cloning of new differentially expressed genes, but also for the study of reassociation kinetics of different DNA in solution, since biotinylated DNA duplexes are specifically and quickly removed from the solution with Ka for the StrAv–Bio complex equal to 10y1 5 M. Thus, the proposed approach of selective enrichment with the required mRNA fractions can be applied to the study of molecular mechanisms of either positive or negative regulation of gene expression in such important biological processes as cell differentiation, embryo development, malignant transformation of cells, and to the study of the action of various stimulators and inhibitors of transcription. The possibility of identifying rare transcripts seems to be particularly important, since the changes in their expression are difficult enough to be determined by standard methods Žincluding the differential display technique, DNA chips..
Acknowledgements The authors thank E. Gorshkova and B. Kondik for the assistance in manuscript preparing. Work was supported by RFBI Grants 99-04-49166, 01-04-48506 and INTAS 99-01200.
References w1x Barr FJ, Emanuel BS. Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA. Anal Biochem 1990;186:369–73. w2x Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science 1993;269:946–51. w3x Cho TJ, Park SS. A simulation of subtractive hybridization. Nucleic Acids Res 1998;26:1440–8. w4x Konietzko U, Kuhl D. A subtractive hybridization method for the enrichment of moderately induced sequences. Nucleic Acids Res 1998;26:1359–61. w5x Zeng J, Gorski RA, Hamer D. Differential cDNA cloning by enzymatic degradation subtraction ŽEDS.. Nucleic Acids Res 1994;22:4381–5. w6x Sive HL, John ST. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Res 1988;16:10937. w7x Wilchek M, Bayer EA. The avidin–biotin complex in bioanalytical applications. Anal Chem 1988;171: 1–32. w8x Forster AC, McInnes JL, Skingle DC, Symons RH. Non-radioactive hybridization probes preparated by the chemical labeling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res 1985;13:745–61.
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w9x Takahashi T, Mitsuda T, Okuda K. An alternative nonradioactive method for labeling DNA using biotin. Anal Biochem 1989;179:77–85. w10x Kohne DE, Levinson SA, Bayers MJ. Room temperature method for the rate of DNA reassociation by many thousandfold: the phenol emulsion reassotiation technique. Biochemistry 1977;16:5329–41. w11x Maniatis T, Fritsch EE, Sambrook J. In: Bayev AA, Skryabin KG, editors. Molecular Cloning: A Laboratory Manual. Moscow, Russian Federation: MIR Publishing House; 1984. p. 1–479. w12x Belyavsky A, Vinogradova T, Rajevsky K. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. Nucleic Acids Res 1989;17:2919–32. w13x Langzidge J, Langzidge P, Berquist PL. Extraction of nucleic acids from agarose gels. Anal Biochem 1980;103:264–71. w14x Zasloff M, Gordon RG, Fenselfeld O. A new method for the purification and identification of covalently closed circular DNA molecules. Nucleic Acids Res 1978;5:1139–52. w15x Wetmur JG. Acceleration of DNA renaturation rates. Biopolymers 1975;14:2517–24. w16x Brunvand MW, Schmidt A, Siebenlist U. Nuclear factors interacting with the mitogen-responsive regulatory region of the interleukin-2. J Biol Chem 1988;263:18904–10. w17x Ullman KS, Northrop JP, Verweij CL, Crabtree GR. Transmission of signals from the T lymphocyte antigene receptor to the genes responsible for cell proliferation and immune function; The Missing Link. Annu Rev Immunol 1990;8:421–52. w18x Rivolta MN, Wilcox ER. A novel and simple methodology to generate subtracted cDNA libraries. Nucleic Acids Res 1995;23:2565–6.
Subtractive hybridization of biotinylated DNA in phenol emulsion Alexandr G. Laman, Sergei G. Kurjukov, Elena V. Bulgakova, Nadezhda N. Anikeeva, Feodor A. Brovko ) Branch of Shemyakin and OÕchinnikoÕ Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russian Federation Received 2 December 1999; received in revised form 3 April 2001; accepted 17 May 2001
Abstract A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique ŽPERT. has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5–1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript ŽIL-2. in three cycles of subtractive hybridization. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Subtractive hybridization; Biotinylated DNA; Differential cDNA libraries; Driver DNA; Tracer DNA; PERT
1. Introduction The methods for creating cDNA libraries on the basis of subtractive hybridization techniques have now become very popular w1–5x. Differential cDNA libraries are a very useful instrument for revealing and studying the genes with the regulated expression. The completeness of DNA hybridization, the hybridization time, and the degree of AbbreÕiations: Bio, biotin; Bio-DNA, biotinylated DNA; ExoIII, exonuclease III; IL-2, interleukin-2; PERT, phenol emulsion reassociation technique; PHA, phytohemagglutinin; StrAv, streptavidin ) Corresponding author. Tel.: q7-095-9252342, q7-0967-730853; fax: q7-0967-790527. E-mail address: [email protected] ŽF.A. Brovko.. 0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 1 7 3 - 7
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removal of homologous sequences from the reaction mixture are of critical importance for the process of subtractive hybridization. Thus, any improvements in hybridization techniques are of great importance for research and diagnostic purposes. Due to the high affinity of streptavidin to biotin, biotinylated DNA hybrids can be quickly removed from the reaction mixture w6x. The stability of streptavidin–biotin complexes w7x enables hybridization in a wide pH range Ž4 to 9. and in the presence of organic solvents. The above factors, as well as the simplicity of the DNA biotinylation procedure, make this approach attractive as applied to the method of subtractive hybridization w8,9x. Phenol emulsion reassociation technique ŽPERT. proposed by Kohne et al. w10x is a quick and convenient method of DNA hybridization in water solutions in wide concentration ranges Ž6 = 10y6 to 6400 mkgrml. that significantly increases the rate of DNA reassociation Ž10–1000 times. as compared with hybridization in a single-phase water system. PERT does not require high DNA concentrations ŽDNA solutions of below 4 mkgrml can be used. and significantly reduces the loss of rare sequences. This is particularly vital for rare transcripts, when it is necessary to obtain an enriched cDNA fraction. Nevertheless, hybridization in phenol emulsion was not used in case of biotinylated DNA due to the formation of water-insoluble aggregates of hybrid DNA w5x. We have revealed that the degree and character of DNA biotinylation influence the formation of Bio-DNA aggregates. This study was aimed to optimize the conditions of insertion of biotin residues into DNA and hybridization of the biotinylated DNA as applied to the method of subtractive hybridization in phenol emulsion. The conditions were selected for the enrichment of rare or developmental phase-specific transcripts in a real biological system to estimate the change in the level of gene expression. The method was approved on a model system of enrichment with rare mRNA sequences Žinduced mRNA of interleukin-2..
2. Materials and methods 2.1. Methods of DNA biotinylation 2.1.1. Photobiotinylation For DNA biotinylation, the plasmid pBluescript KS was digested with EcoRI and labelled with wP32x on 5X cohesive ends. A 2-mkg of linearized and denatured plasmid was biotinylated by photoactivated biotin ŽVector laboratories. according to manufacturer’s instruction. 2.1.2. Biotinylation by filling the ends A 2-mkg of the plasmid pBluescript KS linearized by EcoRI was biotinylated at 37 8C for 10 min in 50 mkl of the buffer for Klenow Fragment in the presence of 100 mkM dATP, dGTP, dCTP, bio-4-dUTP, and 5 U of Klenow Fragment Ž10 mM Tris–HCl, pH 8.0, 10 mM MgCl 2 .. To determine the degree of biotinylation, the reaction was performed in the presence of wa-32Px dATP Ž0.3 mkM..
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2.1.3. Biotinylation after partial DNA hydrolysis with ExoIII A 2-mkg of the linearized plasmid was hydrolyzed in 30 mkl of the buffer for Klenow Fragment using 175 U of ExoIII. The time of hydrolysis was chosen to hydrolyze 20–100 nucleotides. The hydrolyzed plasmid was biotinylated using bio-4dUTP by the method described above. Biotinylated DNA was purified from free nucleotides by passing the sample through a minicolumn with Sephadex G25F. 2.2. Hybridization of the biotinylated DNA to the completely homological DNA in phenol emulsion A 200-ng of the biotinylated plasmid pBluescript KS DNA was dissolved in 45 mkl of TE buffer, and then mixed with 200 ng of 5X-w32Px-labelled DNA of the same plasmid. Then the solution was denaturated by boiling for 2 min, supplemented with 45 mkl of 2.5 M lithium perchlorate and 10 mkl of 90% phenol ŽpH 9.0., and hybridized for 1 h at room temperature and with intensive stirring. On hybridization, the water and phenol phases were separated by centrifugation. Nine acetone volumes were added to the water phase, cooled in ice for 5 min, and centrifuged; the pellet was dissolved in 96 mkl of TE buffer Ž10 mM Tris–HCl, pH 7.5, 1 mM EDTA.. The amount of insoluble DNA aggregates formed during hybridization was determined by measuring the radioactivity of equal aliquots Ž0.1 mkl. of the water phase before and after the hybridization. 2.3. Estimation of hybridization degree and completeness of remoÕal of Bio-DNA hybrids and non-biotinylated DNA On hybridization Žthe same as in Section 2.2., DNA was precipitated with ethanol and dissolved in 100 mkl of SSC Ž1 = . buffer; streptavidin was added to a concentration of 2 mkgrml, and in 30 min the complexes of StrAv–Bio-DNA hybrids were removed using phenol:chloroform Ž1:1. extraction. The percentage of remaining and removed DNA was assessed by the radioactivity ratio of water phases before and after the extraction. 2.4. Synthesis and amplification of cDNA 10 5 cells of the line Jurkat were taken to isolate driver RNA. 10 5 cells of the line Jurkat after their stimulation with phytohemaglutinin by the method described by Belyavsky et al. w12x were taken to isolate tracer RNA. The total RNA was isolated from cells by the guanidinium isothiocyanate method w11x. The tracer and driver cDNA were synthesized using a Universal Riboclone cDNA Synthesis System ŽPromega.. Oligonucleotide adapters B49 Ž5X-AATTCGTCGACATCGAT. and B50 Ž5X-ATCGATGTCGACG. were joined to the double-stranded driver cDNA. Adapters B5 Ž5X-ATTCCTGCAGAATTCTCC., B6 Ž5X GGAGAATTCTGCAGG. were joined to the tracer cDNA. The only one round with 30 cycles of amplification with the primers B49 and B5 was performed for the driver and tracer, respectively, in 100 mkl reaction mixture containing 2 mM MgCl2, 0.3 mkM primer, 0.25 mM dNTP, 1 U of Taq polymerase in the following regime: t 8C: 96–1 min, 46–1 min, 72–2 min on a device THC-2 ŽTechne, England..
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2.5. Procedure of subtractiÕe hybridization The first cycle of subtraction: 200 ng of non-biotinylated tracer DNA and 2 mkg of driver DNA biotinylated using bio-4-dUTP were dissolved in 45 mkl of TE buffer, denatured by boiling for 2 min, and supplied with 45 mkl of 2.5 M lithium perchlorate and 10 mkl of 90% phenol ŽpH 9.0.. Hybridization was carried out for 0.5–1 h at room temperature and with intensive stirring. On hybridization, DNA from the water phase was precipitated with 10 volumes of acetone and centrifuged; the pellet was dissolved in 45 mkl of TE buffer. Streptavidin was added to a concentration of 2 mkgrml and stirred during 10 min, then the complexes of biotinylated DNA–streptavidin were removed by extraction with an equal volume of phenol:chloroform Ž1:1, vrv. and ethanol precipitation. The second cycle of subtraction: 1 mkg of biotinylated driver DNA and lithium perchlorate solution were added to the water phase containing tester DNA to a lithium perchlorate concentration of 1.25 M, and phenol to a concentration of 10%. Hybridization was carried out as in the first cycle. The third and fourth cycles of hybridization proceeded in the same way. 2.6. Hybridization in the presence of dextran sulfate Two hundred nanograms of non-biotinylated w32Px-labelled DNA of the linearized plasmid pBluescript KS and 200 ng of DNA of the same plasmid biotinylated using bio-4-dUTP Žas in 2.1. were dissolved in 20 mkl of water, denatured by heating to 95 8C for 5 min, and then hybridized in 200 mkl of 6 = SSC solution containing 10% dextran sulfate for 1 h at 60 8C w11x. After hybridization, DNA was precipitated and dissolved in a 1 = SSC buffer Ž150 mM NaCl, 15 mM Na citrate, pH 7.4.; streptavidin was added to 2 mkgrml, stirred, and after 10-min incubation, the complexes of protein–Bio-DNA were removed by extraction with: Ž1. phenol:chloroform mixture Ž1:1, vrv., Ž2. chloroform, Ž3. ether. The amount of hybridized DNA was assessed by the ratio of water phase radioactivity level before and after the hybridization. 2.7. Blot hybridization After three cycles of subtraction, the tracer cDNA was amplified as described in Section 2.4. A 1-mkg of each cDNA Žtracer, driver, and subtracted tracer cDNA. was separated in 1.5% agarose gel, transferred to a membrane Hybond C Extra ŽAmersham., and hybridized with w32Px-labelled oligonucleotide specific for cDNA IL-2 dŽAGTGCACCTACTTCAAGT.. Electrophoresis in agarose, transfer and hybridization were carried out according to the standard protocol w11x. 2.8. Calculation of the efficiency of subtraction The zones of hybridization filters of equal areas, corresponding to IL-2 cDNA ŽFig. 2., were taken to determine the degree of enrichment; their radioactivity was assessed by scintillation on a counter LS-6800 ŽBeckman.. The radioactivity of the filter site of the corresponding cDNA of the cells not stimulated by PHA was taken as a base line. The
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factor of enrichment Fe was calculated as a ratio of radioactivities of filter zones by the formula Fe s ŽSub y N .rŽStim y N .. In our case, Sub s 16100" 100, Stims 85 " 10, and N s 30 " 10 and enrichment factor s 292.18. 3. Results and discussion The stage of hybridization is the most time-consuming in the gene engineering experiments on the hybridization of nucleic acids. Therefore, the improvement of hybridization methods is an urgent task. We have proposed a method of subtractive hybridization in phenol emulsion using biotinylated DNA. It has the advantages of PERT, significantly shortening the time of hybridization and increasing the rate of homologous DNA reassociation as compared with the standard hybridization in a single-phase water system. This is particularly important in the work with small amounts of available DNA and in DNA hybridization at low concentrations Žless than 40 mkgrml.. Previously, it was considered impossible to use Bio-DNA in PERT w5x. The method we have developed employs both Bio-DNA in PERT and an efficient technique of duplex DNA removal using the streptavidin–biotin system. At first, we studied the formation of water-insoluble Bio-DNA aggregates under the PERT conditions and analyzed the efficiency of removal of Bio-DNA and their heteroduplexes from the reaction mixture after incubation with streptavidin and phenol extraction. The following conditions appeared to be optimal: 10% phenol Žvrv., pH 9.0, and 1.25 M solution of lithium perchlorate as a chaotropic agent. These conditions were used for hybridization of biotinylated DNA. DNA of the linearized plasmid pBluescript KS with biotin residues joined by different methods was chosen as a model system. Bio-DNA was hybridized under the PERT conditions with the same non-biotinylated DNA but labelled with w32Px by the 5X end. The amount of aggregated DNA was assessed by the change in the DNA content in the water phase after separation of the phenol phase. The results of these experiments are given in Table 1. The data show that not only the degree but also the character of biotinylation are the critical parameters affecting the
Table 1 The effect of DNA biotinylation method on the efficiency of subtractive hybridization in phenol emulsion Method of DNA biotinylation
Degree of biotinylation Žbiotin moleculesr DNA molecules.
% of DNA forming water-insoluble aggregates
% of DNA extracted with phenol:chloroform after binding of streptavidin
Photobiotinylation Filling of DNA partially hydrolyzed by ExoIII X Filling of DNA with 5 extending tetranucleotides
ND 8–17
98 82–90
495 495
1–2
-5
495
ND—not determined, n –number of residues Bio per 1 DNA molecule-10 w8x.
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formation of water-insoluble aggregates. The degree of aggregate formation was negligible only at the insertion of one to two biotin molecules into the 3X end of the DNA molecule, and such DNA could be used for hybridization in phenol emulsion. At increasing the biotinylation degree to 8–17 biotin molecules per 3X end of the DNA molecule, the amount of hybrid DNA remaining in the water phase dramatically decreased. The similar process with a still higher percentage of water-insoluble aggregates was also observed with a photobiotinylated DNA, although this method of biotinylation introduces one biotin residue per 300 bp on average w8x. The size of DNA was about 3000 bp; consequently, the quantity of biotin residues per such a molecule was over 10. At the same time, this DNA formed more water-insoluble aggregates than DNA biotinylated by the 3X end of the molecule, which is evidently due to a statistically random character of the photobiotinylation reaction. We assume that the reduced formation of water-insoluble aggregates at hybridization under the PERT conditions for DNA with one to two biotin residues on 3X ends is explained by biotin distribution along the DNA molecule. It is known that at low pH values Ž- 4.2. w13x, the linear DNA molecules pass to the phenol phase under extraction. Cetyltrimethylammonium salt of DNA is extracted by butanol w14x. In both cases, due either to charge neutralization or to the formation of a salt with the aliphatic base, the hydrophobicity increases over the whole DNA molecule, which is responsible for its passing to the organic phase. We have suggested that the formation of aggregates during hybridization under the PERT conditions is associated with the character of biotin distribution over the DNA molecule. The even distribution of biotin may result in the formation of uniformly hydrophobic molecular structures. Thus, DNA acquires the ability to form associates passing to the organic phase, in particular to phenol. It may be assumed that the smaller the region forming the hydrophobic structure, the lower is its affinity to the organic phase and, consequently, the ability of DNA to pass to the latter. We decided to test this assumption with regard to the fact that the DNA hydrophobicity decreases with the pH increase, using three methods of biotinylation: Ž1. photobiotinylation, providing an even distribution of biotin over the DNA molecule; Ž2. partial hydrolysis of the 3X end of DNA in controlled regions followed by the filling of the ends in the presence of a biotinylated analog of nucleoside triphosphate Žbio-4-dUTP.. In this case, 8 to 17 biotinylated nucleotides were incorporated into DNA; Ž3. the filling of EcoRI-digested DNA by Klenow Fragments and biotinylated bio-4-dUTP, with no more than two biotinylated bases incorporated into DNA molecules. Thus, the insertion of one to two biotin residues into the DNA molecule ends appeared to be the best, since this method of biotinylation does not significantly change the physical and chemical properties of DNA under the PERT conditions. The completeness of hybridization, the degree of removal of heteroduplexes and driver DNA, and the time of performance are the most important factors for subtractive hybridization. The completeness of hybridization was analyzed using a w32Px-labelled DNA from the plasmid pBluescript KS hydrolyzed by EcoRI and the same DNA labelled with biotin by filling the cohesive ends. The quantitative analysis of DNA after hybridization showed that about 95% of plasmid DNA is hybridized in 60 min in case of insertion of one to two biotin molecules into the 3X end.
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At present, the hybridization procedure is accelerated by other widely used methods with enhancers, such as dextran sulfate w15x. For comparison, we have chosen a widespread method of DNA solution hybridization in the presence of 10% dextran sulfate w11x. A plasmid DNA biotinylated by the 3X end and a similar DNA labelled with w32Px by the 5X end were taken as a model system. Our data show that only 37% plasmid DNA was reassociated in 60 min of hybridization in the solution with dextran sulfate, i.e., the Bio-DNA hybridization in phenol emulsion is much more efficient than with the simultaneous addition of dextran sulfate. Besides, it is very difficult to purify the subtracted DNA from dextran sulfate admixtures after hybridization to use it in further experiments on cloning and PCRamplification due to the inhibitory properties of dextran sulfate to many enzymes. The results of plasmid DNA hybridization in phenol emulsion using Bio-DNA served as a basis of approaches to the search and enrichment of rare or phase-specific mRNA. The T-cell lane Jurkat stimulated with phytohemaglutinin w16x was used as a model. With such type of stimulation, new mRNA are synthesized, which are extremely few or completely absent in non-stimulated cells w16,17x. The stimulation of T-cells induces the expression of about 100 genes, one of which Žthe gene of interleukin-2. was chosen as a model w16x. cDNA from stimulated cells Žtracer. were labelled with w32Px, and cDNA from non-stimulated cells Ždriver. were biotinylated. The hybridization of amplified cDNA was performed under the PERT conditions with a 10-fold excess of driver DNA in relation to tracer DNA. The analysis of hybridized DNA showed ŽFig. 1. that about 90% are hybridized in 30 min. The second cycle of hybridization reduced the amount of non-hybridized DNA to
Fig. 1. The efficiency of removal of tracer sequences during subtractive hybridization. Axes are the percentage of tracer DNA in solution and the number of subtractive cycles.
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Fig. 2. Blot hybridization of DNA probes Ž1 mkg each. with interleukin-2 specific probe. Lane 1: P32-labelled DNA mass markers 393 and 270 bp Ž30000 and 40000 cpm per band.. Lane 2: Driver DNA. Lane 3: Non-subtracted tracer. Lane 4: Subtracted and amplified tracer.
2.9%. The third and further cycles did not show any reliably detectable changes in the amount of non-hybridized tracer DNA. The data obtained lead to the following conclusion: a great part of frequent sequences is removed during the first cycle of hybridization. Intermediate and rare DNA are removed during the second and further cycles. Thus, one to two cycles of hybridization under the above described conditions are enough for practical work. The decisive criterion of subtractive hybridization is the degree of enrichment with desired sequences. Three cycles of tracer cDNA subtraction with biotinylated driver cDNA were made to determine the degree of enrichment. The degree of enrichment of tracer cDNA was estimated by the ratio of radioactivity of enriched, stimulated and non-stimulated DNA after blot hybridization with a radioactively labelled oligonucleotide probe to cDNA of interleukin-2 and radioautography ŽFig. 2.. As Fig. 2 shows, the zone corresponding to IL-2 cDNA Žlane 1. is actually absent on lane 2 after an overnight exposure. The analysis of radioactivity of equal-squared filter sections corresponding to the subtracted and stimulated DNA showed a 300-fold efficiency of subtraction. The radioactivity of the same filter section of lane 3 of non-stimulated cells was used as a base level of the above calculation. Rare mRNA, e.g., mRNA of transferin receptor, are enriched 20-fold after four cycles of subtraction as shown by Rivolta and Wilcox w18x. In our case, the degree of enrichment about 300-fold was achieved in three cycles of subtraction.
4. A simplified description of the method of Bio-DNA subtractive hybridization in phenol emulsion and its application We have proposed a method of subtractive hybridization using biotinylated DNA and PERT. The above method reduces the time of reassociation from the conventional 24–48 h to 0.5–1 h at the tracer DNA concentration of 4 mkgrml. DNA heterohybrids are removed by a highly efficient method of Bio-DNA extraction as complexes with
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streptavidin. Using different adapter pairs to obtain tracer and driver, DNA permits a selective amplification of the tracer upon the last cycle of hybridization. Besides, the completeness of the driver DNA removal prevents the non-specific amplification and contamination of subtracted DNA. A possibility of high-degree enrichment for rare mRNA sequences was shown in the model system of IL-2 mRNA. The proposed technique does not require high DNA concentrations; the subtractive hybridization can proceed with the volumes commonly used in biochemical practice and at room temperature. The proposed method can be used not only for the cloning of new differentially expressed genes, but also for the study of reassociation kinetics of different DNA in solution, since biotinylated DNA duplexes are specifically and quickly removed from the solution with Ka for the StrAv–Bio complex equal to 10y1 5 M. Thus, the proposed approach of selective enrichment with the required mRNA fractions can be applied to the study of molecular mechanisms of either positive or negative regulation of gene expression in such important biological processes as cell differentiation, embryo development, malignant transformation of cells, and to the study of the action of various stimulators and inhibitors of transcription. The possibility of identifying rare transcripts seems to be particularly important, since the changes in their expression are difficult enough to be determined by standard methods Žincluding the differential display technique, DNA chips..
Acknowledgements The authors thank E. Gorshkova and B. Kondik for the assistance in manuscript preparing. Work was supported by RFBI Grants 99-04-49166, 01-04-48506 and INTAS 99-01200.
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