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Determination of DNA base composition by small scale acrylamide–CsCl gradient centrifugation
J. Biochem. Biophys. Methods 63 (2005) 155 – 160 www.elsevier.com/locate/jbbm
Determination of DNA base composition by small scale acrylamide–CsCl gradient centrifugation Il-Young Ahn, Carlos E. Winter* Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 1374, 05508-900 Sa˜o Paulo, Brazil Received 13 September 2004; accepted 22 March 2005
Abstract In this paper we describe a simple and rapid protocol for DNA base composition determination by CsCl gradient in the presence of acrylamide. This method permits the determination of GC content in microgram amounts of DNA, and results are easily documented in photographs or graphs. The protocol was applied to the characterization of nematode DNA, but can be used for other organisms. Analyzing several experiments the mean standard deviation observed in the calculated GC content is near 1.3%. D 2005 Elsevier B.V. All rights reserved. Keywords: CsCl gradient; DNA; GC content; Ultracentrifugation; Polyacrylamide
1. Introduction The physico-chemical characterization of DNA is an important step for further studies on the genome of different organisms. For example, before we begin a genome project it is advisable to have an idea of the size and complexity of the haploid content of DNA in the cells of the organism under study [1]. Since the 1960s several analytical approaches have been developed to obtain the necessary data [2]. The analysis of
* Corresponding author. Tel.: +55 11 3091 7269; fax: +55 11 3091 7417. E-mail address: [email protected] (C.E. Winter). 0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2005.03.002
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I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
several genomes have shown that gene density, gene length, and patterns of codon usage as well as the distribution of different classes of repetitive elements are all related to GC content [3–5]. GC content determination is also indispensable for further kinetic characterization of the hereditary material, using Cot curves. Recently Peterson proposed a strategy for plant genome analysis based on Cot curves [6] that can be applied also to other organisms [7]. Several studies have been done to determine the base composition (%GC) of genomic DNA to obtain primary data for sequencing projects. Base composition can be determined through analytical ultracentrifugation in CsCl gradients [8]. This method is accurate and reproducible; however, if an analytical ultracentrifuge is not available, cumbersome protocols have been described for thermal denaturation analysis [9]. Caesium chloride gradients can be generated in the presence of acrylamide and riboflavin and DNA bands in the gradient can be fixed after light induced polymerization [10]. This protocol does not involve inconvenient gradient fractionation and permits direct measurement of DNA band position. Preston et al. [10] detected the DNA bands with dStains-allT [11], a non-fluorescent dye for biological macromolecules. However, their protocol was time-consuming, causing swelling of the gels and distortion of the bands. Here we report a modified protocol using Ethidium Bromide (EtBr) and a device for the rapid destaining of the gels. This protocol permits the rapid and precise determination of GC content using small amounts of DNA. The accuracy of this technique for determination of the GC content of DNA samples was shown to be as good as that obtained by Tm curves, if internal standards are run with the unknown DNA. This approach was successfully applied to the determination of GC content of the nematode, Oscheius tipulae, strain CEW1, a sister species of Caenorhabditis elegans [12].
2. Materials and methods 2.1. DNA density markers and extraction Three DNA samples of known density were used as markers: Escherichia coli HB101 (GC = 50%) [8], Micrococcus luteus (GC = 72%) [8] and Caenorhabditis elegans N2 (GC = 35.5%) [18]. C. elegans cultures were kindly provided by the Caenorhabditis Genetics Center (University of Minnesota). Genomic DNA was purified from exponential cultures of E. coli and M. luteus as described in [13]. DNA from two nematodes, C. elegans and Oscheius tipulae CEW1 (a strain isolated and maintained at the Department of Parasitology, University of Sa˜o Paulo) were purified as follows. The worms were grown as previously described [14]. Prior to DNA extraction, worms from asynchronous cultures were incubated at 22 8C in S buffer (50 mM potassium phosphate, 100 mM sodium chloride, pH 6.0) for 2 h. During this time, bacteria within the intestine were digested, eliminating possible bacterial DNA contamination. Worm genomic DNA was then extracted as described by Sulston and Hodgkin [15]. Purity of the worm DNA samples was established after PCR, using primers for E. coli rRNA genes. Only samples that showed no amplification products were used. DNA preparations were sheared at 4 8C with syringe and needle (30G 1/2 in.; 0.3 13 mm) and purified with Chelex-100 resin
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
157
(BioRad). DNA fragments obtained ranged from 3 to 7 kbp, and DNA concentrations were determined by densitometry of electrophoresis in 1% agarose gel. 2.2. Preparation of CsCl gradients centrifugation with acrylamide The initial density was adjusted to get the bands of DNA samples in the middle of the tube after centrifugation. Density of CsCl 65% (w/v) stock solution was checked by refractometry and adjusted to 1.9052 g/cm3 [16]. CsCl gradient centrifugations were done with 1–2 Ag of sheared genomic DNA dissolved in a solution of CsCl at the initial density of 1.7248 g/cm3, containing 7.5% (w/v) acrylamide, 0.38% (w/v) bis-acrylamide, 0.16% (v/v) TEMED, 10 mM Tris pH 8.0, 1 mM EDTA in a total volume of 1831 Al. DNA solution was applied to a bQuik-SealkQ Polyallomer tube (Beckman; Cat Nr. 344635). In a dark room, an aliquot of a saturated solution of riboflavin (approximately 1 mg/ml), previously filtered in a 0.22 Am filter [10], was added to the DNA solution to obtain a final volume of 1840 Al. Mineral oil was added to fill up the tube. Tubes were sealed by melting and centrifuged in an Optimak mini-ultracentrifuge (Beckman) at 156,844 g for 40 h at 20 8C in a TLA 120.2 rotor (Beckman). Centrifugations done in less than 38 h resulted in fuzzy DNA bands. Immediately after centrifugation, acrylamide monomers were polymerized by exposure of the centrifuge tubes to a fluorescent light (5 cm from a 15 W daylight lamp) for 10 min. The tubes were cut at the top and the polyacrylamide gels were extruded by pressing the bottom, stained for 10 min in a solution of EtBr (0.5 Ag/ml) and excess stain was removed from the gel by washing for 2–3 h in the destaining device (Fig. 1A) constructed with a disposable 50-ml plastic centrifuge tube perforated with a hot nail. Distilled water was circulated through the destaining device at a flux of 810 ml per min using a peristaltic pump. The water was continuously filtered through activated charcoal to speed up the destaining process and diminish contamination with ethidium bromide. DNA bands were visualized in a UV transilluminator and photographed with a CCD camera (EagleEye II, Stratagene). 2.3. Analysis of gradients Photographs of stained gels were analyzed using the BandLeader software [17]. The density profiles were plotted and analyzed using Excel (Microsoft, version 9.0) and Origin 4.10 (Microcal, Northampton). The relative distance of DNA standard bands in the tube was plotted against the known GC content of the markers and the standard curve was obtained by least squares linear regression using Origin 4.10. Using this standard curve, floating density of DNA samples could be determined with the formula: q = (%GC 0.098) + 1.660qinffiffiffiffiffiffiffiffiffiffiffiffiffi whichffi 2 3 x2 U is the density in g/cm [8]. Standard deviation (S.D.) was obtained from S:D: ¼ Rx n¯ , n when 65% of data will fall within one standard deviation. 2.4. Thermal denaturation curve Tm determinations were done using a spectrophotometer GBC UV–visible specially adapted to this end. The DNA samples were denatured by boiling in 0.1X SSC at a final concentration of 20 ng/Al in a total volume of 1 ml. The cuvette temperature was maintained
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Fig. 1. Small scale CsCl gradient ultracentrifugation. (A) Schematic representation of the destaining device. (B) Densitometric tracings of Ethidium Bromide stained gels. DNA density markers used were: M: Micrococcus luteus (1.731 g/cm3); E: Escherichia coli strain HB101 (1.710 g/cm3) and C: Caenorhabditis elegans (strain N2) (1.695 g/cm3). O: Oscheius tipulae (strain CEW1). The bands were aligned using the position of the M. luteus DNA band. The dotted and continuous lines in the graph correspond, respectively, to the top and bottom gels shown in the inset. (C) Standard curve of the %GC markers, used to calculate the density of O. tipulae genomic DNA. The linear regression equation is shown. Data shown in panels (B) and (C) were obtained in a single representative experiment.
at 60 8C for 10 min and then slowly increased to 97 8C at a rate of 1.5 8C per min. The absorbance at 260.0 nm was monitored continuously. The Tm was determined as the first derivative of the melting curve using the program DNA Melt V1.6 (GBC 916). The GC content was calculated as described using the equation: GC (%) = 2.44 {Tm 81.5 16.6 log (M)} (M = 0.0150), where M is the Na+ concentration in mol/L [9].
3. Results and discussion Fig. 1B and C shows representative results obtained using the protocol here described. Using ethidium bromide to stain the DNA bands, instead of dStains-allT
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
159
Table 1 Determination of GC content of Oscheius tipulae DNA by melting curves and acrylamide–CsCl density gradients Tm
O. tipulae
CsCl–acrylamide gradient
Temperature (8C) (mean F S.D.) (n = 5)
GC (%) (mean F S.D.) (n = 5)
Density (g/cm3) (mean F S.D.) (n = 5)
GC (%) (mean F S.D.) (n = 5)
68.40 F 0.10
41.90 F 0.24
1.7024 F 0.001
43.28 F 0.63
[11], and the destaining device shown here (Fig. 1A) we reduced the processing time of gels after centrifugation from 18 h [10] to 3 h. With this simple alteration in the protocol we could obtain sharper DNA bands without swelling or distorting the gel (Fig. 1B). The density tracing of a representative gel is shown in Fig. 1B. Buoyant density of unknown samples can be calculated using standard curves as the one shown in Fig. 1C. Using a standard curve, the %GC of O. tipulae genomic DNA was determined as 43.3, which is equivalent to a density of 1.7024 g/cm3 (Table 1). This is almost the same GC content determined using a Tm curve (41.9%). The difference between the two methods was approximately 1.4% GC content. This error is almost the same described previously [10]. Including the centrifugation time, the whole process could be done in less than three working days. Using this small scale CsCl centrifugation only 2 Ag of genomic DNA is required instead of 20 Ag used for Tm determination.
4. Simplified description of the method and its applications In the present paper we describe a modified protocol for the determination of DNA GC content using ultracentrifugation. This protocol allows the characterization of several DNA samples in a single run, is easy to establish, does not need highly purified DNA samples, and gives reproducible results. As little as 2 Ag of DNA are enough to obtain highly reproducible results. CsCl–acrylamide DNA ultracentrifugation should be useful as a first step in the characterization of procaryote or eukaryote genomes. A detailed protocol will be sent upon request (Appendix).
Acknowledgements We thank Dr Arthur Gruber from the Department of Pathology, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, for spectrophotometer facilities, Drs A. Tania Bijovsky and Silvia R. Uliana from the Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, for critical reading of an early version of the manuscript and Dr Jeffrey Jon Shaw from the Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo for critical reading of the final version. IYA is a graduate fellow from FAPESP (The State of Sa˜o Paulo Research Foundation). This work was supported by FAPESP.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbbm.2005.03.002.
References [1] Lewis R. Cot analysis stages a revival — A three-decade-old technique finds new life in the genomicist’s tool chest. Scientist 2002;16:49 [http://www.the-scientist.com/yr2002/aug/lcprofile2_020819.html]. [2] Goldberg RB. From cot curves to genomics — how gene cloning established new concepts in plant biology. Plant Physiol 2001;125:4 – 8. [3] Zoubak S, Clay O, Bernardi G. The gene distribution of the human genome. Gene 1996;174:95 – 102. [4] Duret L, Mouchiroud D, Gautier C. Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores. J Mol Evol 1995;40:308 – 17. [5] Sharp PM, Averof M, Lloyd AT, Matassi G, Peden JF. DNA sequence evolution: the sounds of silence. Philos Trans R Soc Lond Biol Sci 1995;349:241 – 7. [6] Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, et al. Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. Genome Res 2002;12:795 – 807. [7] Wicker T, Robertson JS, Schulze SR, Feltus FA, Magrini V, Morrison JA, et al. The repetitive landscape of the chicken genome. Genome Res 2004;15:126 – 36. [8] Schildkraut CL, Marmur J, Doty P. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J Mol Biol 1962;4:430 – 43. [9] Marmur J, Doty P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 1962;5:109 – 18. [10] Preston JF, Boone DR. Analytical determination of the buoyant density of DNA in acrylamide gels after preparative CsCl gradient centrifugation. FEBS Lett 1973;37:321 – 4. [11] Dahlberg AE, Dingman CW, Peacock AC. Electrophoretic characterization of bacterial polyribosomes in agarose–acrylamide composite gels. J Mol Biol 1969;41:139 – 47. [12] Winter CE. The yolk polypeptides of a free living rhabditid nematode. Comp Biochem Physiol 1992;103B:189 – 96. [13] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Plainview (NY)7 Cold Spring Harbor Lab. Press; 1989. [14] Sulston JE, Brenner S. The DNA of Caenorhabditis elegans. Genetics 1974;77:95 – 104. [15] Sulston JE, Hodgkin J. In: Wood WB, editor. Community of C. elegans researchers, editors. The nematode Caenorhabditis elegans. Cold Spring Harbor7 Cold Spring Harbor Laboratory Press; 1988. p. 587 – 606. [16] Flamm WG, Birnstiel ML, Walker PMB. Preparation and fractionation, and isolation of single strands of DNA by isopycnic ultracentrifugation in fixed-angle rotors. In: Birnie GD, Fox SM, editors. Subcellular components, preparation and fractionation. London7 Butterworth and Co; 1968. p. 125 – 55. [17] Aharoni, M. Band Leader version 1.0 — available at http://iubio.bio.indiana.edu/soft/molbio/ibmpc/ bandleader.exe; 1993. [18] Stein LD, Bao Z, Blasiar D, Blumenthal T, Brent MR, Chen N, et al. The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics. PLoS Biol 2003;1:166 – 92.
Determination of DNA base composition by small scale acrylamide–CsCl gradient centrifugation Il-Young Ahn, Carlos E. Winter* Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 1374, 05508-900 Sa˜o Paulo, Brazil Received 13 September 2004; accepted 22 March 2005
Abstract In this paper we describe a simple and rapid protocol for DNA base composition determination by CsCl gradient in the presence of acrylamide. This method permits the determination of GC content in microgram amounts of DNA, and results are easily documented in photographs or graphs. The protocol was applied to the characterization of nematode DNA, but can be used for other organisms. Analyzing several experiments the mean standard deviation observed in the calculated GC content is near 1.3%. D 2005 Elsevier B.V. All rights reserved. Keywords: CsCl gradient; DNA; GC content; Ultracentrifugation; Polyacrylamide
1. Introduction The physico-chemical characterization of DNA is an important step for further studies on the genome of different organisms. For example, before we begin a genome project it is advisable to have an idea of the size and complexity of the haploid content of DNA in the cells of the organism under study [1]. Since the 1960s several analytical approaches have been developed to obtain the necessary data [2]. The analysis of
* Corresponding author. Tel.: +55 11 3091 7269; fax: +55 11 3091 7417. E-mail address: [email protected] (C.E. Winter). 0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2005.03.002
156
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
several genomes have shown that gene density, gene length, and patterns of codon usage as well as the distribution of different classes of repetitive elements are all related to GC content [3–5]. GC content determination is also indispensable for further kinetic characterization of the hereditary material, using Cot curves. Recently Peterson proposed a strategy for plant genome analysis based on Cot curves [6] that can be applied also to other organisms [7]. Several studies have been done to determine the base composition (%GC) of genomic DNA to obtain primary data for sequencing projects. Base composition can be determined through analytical ultracentrifugation in CsCl gradients [8]. This method is accurate and reproducible; however, if an analytical ultracentrifuge is not available, cumbersome protocols have been described for thermal denaturation analysis [9]. Caesium chloride gradients can be generated in the presence of acrylamide and riboflavin and DNA bands in the gradient can be fixed after light induced polymerization [10]. This protocol does not involve inconvenient gradient fractionation and permits direct measurement of DNA band position. Preston et al. [10] detected the DNA bands with dStains-allT [11], a non-fluorescent dye for biological macromolecules. However, their protocol was time-consuming, causing swelling of the gels and distortion of the bands. Here we report a modified protocol using Ethidium Bromide (EtBr) and a device for the rapid destaining of the gels. This protocol permits the rapid and precise determination of GC content using small amounts of DNA. The accuracy of this technique for determination of the GC content of DNA samples was shown to be as good as that obtained by Tm curves, if internal standards are run with the unknown DNA. This approach was successfully applied to the determination of GC content of the nematode, Oscheius tipulae, strain CEW1, a sister species of Caenorhabditis elegans [12].
2. Materials and methods 2.1. DNA density markers and extraction Three DNA samples of known density were used as markers: Escherichia coli HB101 (GC = 50%) [8], Micrococcus luteus (GC = 72%) [8] and Caenorhabditis elegans N2 (GC = 35.5%) [18]. C. elegans cultures were kindly provided by the Caenorhabditis Genetics Center (University of Minnesota). Genomic DNA was purified from exponential cultures of E. coli and M. luteus as described in [13]. DNA from two nematodes, C. elegans and Oscheius tipulae CEW1 (a strain isolated and maintained at the Department of Parasitology, University of Sa˜o Paulo) were purified as follows. The worms were grown as previously described [14]. Prior to DNA extraction, worms from asynchronous cultures were incubated at 22 8C in S buffer (50 mM potassium phosphate, 100 mM sodium chloride, pH 6.0) for 2 h. During this time, bacteria within the intestine were digested, eliminating possible bacterial DNA contamination. Worm genomic DNA was then extracted as described by Sulston and Hodgkin [15]. Purity of the worm DNA samples was established after PCR, using primers for E. coli rRNA genes. Only samples that showed no amplification products were used. DNA preparations were sheared at 4 8C with syringe and needle (30G 1/2 in.; 0.3 13 mm) and purified with Chelex-100 resin
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
157
(BioRad). DNA fragments obtained ranged from 3 to 7 kbp, and DNA concentrations were determined by densitometry of electrophoresis in 1% agarose gel. 2.2. Preparation of CsCl gradients centrifugation with acrylamide The initial density was adjusted to get the bands of DNA samples in the middle of the tube after centrifugation. Density of CsCl 65% (w/v) stock solution was checked by refractometry and adjusted to 1.9052 g/cm3 [16]. CsCl gradient centrifugations were done with 1–2 Ag of sheared genomic DNA dissolved in a solution of CsCl at the initial density of 1.7248 g/cm3, containing 7.5% (w/v) acrylamide, 0.38% (w/v) bis-acrylamide, 0.16% (v/v) TEMED, 10 mM Tris pH 8.0, 1 mM EDTA in a total volume of 1831 Al. DNA solution was applied to a bQuik-SealkQ Polyallomer tube (Beckman; Cat Nr. 344635). In a dark room, an aliquot of a saturated solution of riboflavin (approximately 1 mg/ml), previously filtered in a 0.22 Am filter [10], was added to the DNA solution to obtain a final volume of 1840 Al. Mineral oil was added to fill up the tube. Tubes were sealed by melting and centrifuged in an Optimak mini-ultracentrifuge (Beckman) at 156,844 g for 40 h at 20 8C in a TLA 120.2 rotor (Beckman). Centrifugations done in less than 38 h resulted in fuzzy DNA bands. Immediately after centrifugation, acrylamide monomers were polymerized by exposure of the centrifuge tubes to a fluorescent light (5 cm from a 15 W daylight lamp) for 10 min. The tubes were cut at the top and the polyacrylamide gels were extruded by pressing the bottom, stained for 10 min in a solution of EtBr (0.5 Ag/ml) and excess stain was removed from the gel by washing for 2–3 h in the destaining device (Fig. 1A) constructed with a disposable 50-ml plastic centrifuge tube perforated with a hot nail. Distilled water was circulated through the destaining device at a flux of 810 ml per min using a peristaltic pump. The water was continuously filtered through activated charcoal to speed up the destaining process and diminish contamination with ethidium bromide. DNA bands were visualized in a UV transilluminator and photographed with a CCD camera (EagleEye II, Stratagene). 2.3. Analysis of gradients Photographs of stained gels were analyzed using the BandLeader software [17]. The density profiles were plotted and analyzed using Excel (Microsoft, version 9.0) and Origin 4.10 (Microcal, Northampton). The relative distance of DNA standard bands in the tube was plotted against the known GC content of the markers and the standard curve was obtained by least squares linear regression using Origin 4.10. Using this standard curve, floating density of DNA samples could be determined with the formula: q = (%GC 0.098) + 1.660qinffiffiffiffiffiffiffiffiffiffiffiffiffi whichffi 2 3 x2 U is the density in g/cm [8]. Standard deviation (S.D.) was obtained from S:D: ¼ Rx n¯ , n when 65% of data will fall within one standard deviation. 2.4. Thermal denaturation curve Tm determinations were done using a spectrophotometer GBC UV–visible specially adapted to this end. The DNA samples were denatured by boiling in 0.1X SSC at a final concentration of 20 ng/Al in a total volume of 1 ml. The cuvette temperature was maintained
158
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
Fig. 1. Small scale CsCl gradient ultracentrifugation. (A) Schematic representation of the destaining device. (B) Densitometric tracings of Ethidium Bromide stained gels. DNA density markers used were: M: Micrococcus luteus (1.731 g/cm3); E: Escherichia coli strain HB101 (1.710 g/cm3) and C: Caenorhabditis elegans (strain N2) (1.695 g/cm3). O: Oscheius tipulae (strain CEW1). The bands were aligned using the position of the M. luteus DNA band. The dotted and continuous lines in the graph correspond, respectively, to the top and bottom gels shown in the inset. (C) Standard curve of the %GC markers, used to calculate the density of O. tipulae genomic DNA. The linear regression equation is shown. Data shown in panels (B) and (C) were obtained in a single representative experiment.
at 60 8C for 10 min and then slowly increased to 97 8C at a rate of 1.5 8C per min. The absorbance at 260.0 nm was monitored continuously. The Tm was determined as the first derivative of the melting curve using the program DNA Melt V1.6 (GBC 916). The GC content was calculated as described using the equation: GC (%) = 2.44 {Tm 81.5 16.6 log (M)} (M = 0.0150), where M is the Na+ concentration in mol/L [9].
3. Results and discussion Fig. 1B and C shows representative results obtained using the protocol here described. Using ethidium bromide to stain the DNA bands, instead of dStains-allT
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
159
Table 1 Determination of GC content of Oscheius tipulae DNA by melting curves and acrylamide–CsCl density gradients Tm
O. tipulae
CsCl–acrylamide gradient
Temperature (8C) (mean F S.D.) (n = 5)
GC (%) (mean F S.D.) (n = 5)
Density (g/cm3) (mean F S.D.) (n = 5)
GC (%) (mean F S.D.) (n = 5)
68.40 F 0.10
41.90 F 0.24
1.7024 F 0.001
43.28 F 0.63
[11], and the destaining device shown here (Fig. 1A) we reduced the processing time of gels after centrifugation from 18 h [10] to 3 h. With this simple alteration in the protocol we could obtain sharper DNA bands without swelling or distorting the gel (Fig. 1B). The density tracing of a representative gel is shown in Fig. 1B. Buoyant density of unknown samples can be calculated using standard curves as the one shown in Fig. 1C. Using a standard curve, the %GC of O. tipulae genomic DNA was determined as 43.3, which is equivalent to a density of 1.7024 g/cm3 (Table 1). This is almost the same GC content determined using a Tm curve (41.9%). The difference between the two methods was approximately 1.4% GC content. This error is almost the same described previously [10]. Including the centrifugation time, the whole process could be done in less than three working days. Using this small scale CsCl centrifugation only 2 Ag of genomic DNA is required instead of 20 Ag used for Tm determination.
4. Simplified description of the method and its applications In the present paper we describe a modified protocol for the determination of DNA GC content using ultracentrifugation. This protocol allows the characterization of several DNA samples in a single run, is easy to establish, does not need highly purified DNA samples, and gives reproducible results. As little as 2 Ag of DNA are enough to obtain highly reproducible results. CsCl–acrylamide DNA ultracentrifugation should be useful as a first step in the characterization of procaryote or eukaryote genomes. A detailed protocol will be sent upon request (Appendix).
Acknowledgements We thank Dr Arthur Gruber from the Department of Pathology, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, for spectrophotometer facilities, Drs A. Tania Bijovsky and Silvia R. Uliana from the Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, for critical reading of an early version of the manuscript and Dr Jeffrey Jon Shaw from the Department of Parasitology, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo for critical reading of the final version. IYA is a graduate fellow from FAPESP (The State of Sa˜o Paulo Research Foundation). This work was supported by FAPESP.
160
I.-Y. Ahn, C.E. Winter / J. Biochem. Biophys. Methods 63 (2005) 155–160
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbbm.2005.03.002.
References [1] Lewis R. Cot analysis stages a revival — A three-decade-old technique finds new life in the genomicist’s tool chest. Scientist 2002;16:49 [http://www.the-scientist.com/yr2002/aug/lcprofile2_020819.html]. [2] Goldberg RB. From cot curves to genomics — how gene cloning established new concepts in plant biology. Plant Physiol 2001;125:4 – 8. [3] Zoubak S, Clay O, Bernardi G. The gene distribution of the human genome. Gene 1996;174:95 – 102. [4] Duret L, Mouchiroud D, Gautier C. Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores. J Mol Evol 1995;40:308 – 17. [5] Sharp PM, Averof M, Lloyd AT, Matassi G, Peden JF. DNA sequence evolution: the sounds of silence. Philos Trans R Soc Lond Biol Sci 1995;349:241 – 7. [6] Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, et al. Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. Genome Res 2002;12:795 – 807. [7] Wicker T, Robertson JS, Schulze SR, Feltus FA, Magrini V, Morrison JA, et al. The repetitive landscape of the chicken genome. Genome Res 2004;15:126 – 36. [8] Schildkraut CL, Marmur J, Doty P. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J Mol Biol 1962;4:430 – 43. [9] Marmur J, Doty P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 1962;5:109 – 18. [10] Preston JF, Boone DR. Analytical determination of the buoyant density of DNA in acrylamide gels after preparative CsCl gradient centrifugation. FEBS Lett 1973;37:321 – 4. [11] Dahlberg AE, Dingman CW, Peacock AC. Electrophoretic characterization of bacterial polyribosomes in agarose–acrylamide composite gels. J Mol Biol 1969;41:139 – 47. [12] Winter CE. The yolk polypeptides of a free living rhabditid nematode. Comp Biochem Physiol 1992;103B:189 – 96. [13] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Plainview (NY)7 Cold Spring Harbor Lab. Press; 1989. [14] Sulston JE, Brenner S. The DNA of Caenorhabditis elegans. Genetics 1974;77:95 – 104. [15] Sulston JE, Hodgkin J. In: Wood WB, editor. Community of C. elegans researchers, editors. The nematode Caenorhabditis elegans. Cold Spring Harbor7 Cold Spring Harbor Laboratory Press; 1988. p. 587 – 606. [16] Flamm WG, Birnstiel ML, Walker PMB. Preparation and fractionation, and isolation of single strands of DNA by isopycnic ultracentrifugation in fixed-angle rotors. In: Birnie GD, Fox SM, editors. Subcellular components, preparation and fractionation. London7 Butterworth and Co; 1968. p. 125 – 55. [17] Aharoni, M. Band Leader version 1.0 — available at http://iubio.bio.indiana.edu/soft/molbio/ibmpc/ bandleader.exe; 1993. [18] Stein LD, Bao Z, Blasiar D, Blumenthal T, Brent MR, Chen N, et al. The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics. PLoS Biol 2003;1:166 – 92.