ANALYTICAL SCIENCES JUNE 2005, VOL. 21 619 2005 © The Japan Society for Analytical Chemistry

Prestaining Method as a Useful Tool for the Agarose Gel Electrophoretic Detection of Polymerase Chain Reaction Products with a Fluorescent Dye SYBR® Gold Nucleic Acid Gel Stain

Emi SUENAGA and Hiroshi NAKAMURA†

Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278–8510, Japan

Three staining methods using SYBR® Gold Nucleic Acid Gel Stain (SYBR Gold) as a fluorescent dye were evaluated for the agarose gel electrophoretic detection of DNA. The methods involve prestain, in-gel stain, and poststain methods. DNA markers and polymerase chain reaction (PCR) products obtained by minisatellite variant repeat-PCR (MVR-PCR) amplification in a D1S8 locus were used as model DNA and practical samples, respectively. Among the three methods tested under the usual electrophoretic conditions, a prestain method using a 10000-fold diluted SYBR Gold solution showed most excellent features regarding cost and rapidity to use with good stainability and resolution over loaded DNA amounts of about 98 ng to 300 ng. The prestain method was found to be applicable to the analysis of DNA in MVR-PCR products from a human hair root.

(Received September 24, 2004; Accepted March 14, 2005)

DNA bands. The excitation spectra of SYBR Introduction Gold-DNA complexes are known to show two excitation maxima at 300 nm and 495 nm, and the fluorescence intensities Various nucleic acid gel stains have been developed so far. of the complexes excitated at 495 nm are 8-times higher than SYBR® Gold Nucleic Acid Gel Stain (SYBR Gold), an those excitated at 300 nm.1 Therefore, if excited with a unsymmetrical cyaninedye reported by Tuma et al.1 in 1999, is wavelength at around 495 nm, more sensitive detection of most sensitive compared to other previously used reagents,1 prestained DNA seems to be carried out even when a diluted such as , SYBR® Green I Nucleic Acid Gel SYBR Gold solution, dilute enough to prevent the migration Stain and SYBR® Green II RNA Gel Stain for detecting double- distraction, was used. Based on the above consideration, we stranded DNA (dsDNA), single-stranded DNA (ssDNA) and aimed at establishing a sensitive SYBR Gold prestain method RNA, though SYBR Gold is more expensive than traditional for the agarose gel electrophoretic detection of DNA by intercalators, such as ethidium bromide. To stain DNA on employing an imaging analyzer equipped with a SHG laser (473 electrophoretic gel with these dyes, there are three methods, nm light). including prestain, in-gel stain, and poststain methods.2 The The method was applied to the analysis of minisatellite variant prestain method has some advantages in that it can be practiced repeat polymerase chain reaction (MVR-PCR) products for with smaller amounts of staining dyes, and does not need any individual identification, and led to the development of a time-consuming poststain process. However, the method has simplified and cost-effective MVR-PCR procedure consisting of some disadvantages, in that the mobility of DNA-dye non-isotope labeling and non-blotting procees, by which complexes is affected by some factors, such as the dye:DNA individual identification could be achieved using a hair root. concentration ratio3–6 and the dye-DNA (bp) ratio.5–7 MVR-PCR, originally reported by Jeffreys et al.,8 was applied A caution has been reported by a SYBR Gold supplier,2 i.e. in the present work to the hypervariable human minisatellite prestaining and in-gel staining with SYBR Gold should be D1S8, which contains two major classes of variant 29 bp repeat avoided because band deformation occurs upon electrophoresis, units, designated as a-type and t-type, which differ in only a leading to a lower resolution; the use of poststain is single base with each other.9,10 Individual identification was recommended. On the other hand, Tuma et al.1 reported that performed by analyzing the individual variation of the 29000-fold or more of diluted SYBR Gold solutions must be combined pattern of a-type and t-type variants in the locus. used for the precise agarose gel electrophoretic separation of This was experimentally achieved by judging from the 120 ng of DNA prestained with SYBR Gold; otherwise, the individual distinction of electrophoretic profiles of each rung migration times of the complexes would be affected by the that appeared in the a-type and t-type ladders with 29 bp reagent concentration. However, the use of such diluted SYBR periodicity. Gold solutions caused a shortage of the reagent to fail to detect

† To whom correspondence should be addressed. 620 ANALYTICAL SCIENCES JUNE 2005, VOL. 21

Experimental

Materials Chelex® 100 was purchased from BIORAD (Richmond, CA, USA). The primers8 for PCR were prepared by QIAGEN (Tokyo, Japan). Marker 4 (φX174/HaeIII digest), Marker 5 (φX174/HincII digest), Marker 11 (pUC19/MspI digest) and Agarose 21 for electrophoresis were obtained from NIPPON GENE (Toyama, Japan). SYBR Gold as a staining reagent was purchased from Molecular Probes (Eugene, OR, USA) and used after 10000-fold or appropriate dilution, as described in the text with a TBE buffer consisting of 89 mM Tris, 89 mM borate, and 2 mM EDTA·2Na at pH 8.3. Water was purified using a Milli- Q system (Millipore; Bedford, MA, USA). The other reagents and buffers used were of guaranteed grade.

DNA extraction DNA was extracted from human hair root by the Chelex method.11,12 The yield of extracted DNA was calculated with the absorbance at 260 nm. In each MVR-PCR reaction, 100 ng of extracted DNA was used.

MVR-PCR The reaction conditions reported by Jeffreys et al.8 were slightly modified as follows. In this method, 20-fold concentrations of both primers 32-TAG-A and 32-TAG-T were used. Samples of genomic DNA were amplified in 50 µL of a PCR solution including 1 µM primer 32D (5′-CGACGCGCAG- ATGGAGCAAT-GGCC-3′), 1 µM primer TAG (5′- TCATGCGTCCATGGTCCGGA-3′) and either 0.2 µM 32- Fig. 1 Comparison of the three staining methods for the detection of DNA markers: the prestain method, the in-gel stain method, and TAG-A (5′-TCATGCGTCCATGGTCCGGACATTCTGAGTC- ′ the poststain method. Asterisk in Marker 4 shows the DNA bands of ACCCCTGGC-3 ) for the amplification of the a-type unit or 0.4 281 bp and 271 bp. Electrophoresis mode, constant voltage (60 V); µ ′ M 32-TAG-T (5 -TCATGCGTCCATGGTCCGGACATTCT- gel, 3% Agarose 21; buffer, TBE buffer; samples, Marker 4 (lane GAGTCACCCCTGGT-3′) for the amplification of the t-type M4), Marker 5 (lane M5) and Marker 11 (lane M11); SYBR Gold unit, plus 2.5 unit Gene Taq DNA polymerase (NIPPON concentration, 10000-fold dilution. The total amount of Marker GENE). Reactions were performed at 94˚C for 1.3 min, at 68˚C loaded per lane was 300 ng. for 1 min, and at 70˚C for 5 min for 30 cycles, followed by incubating at 68˚C for 1 min and at 70˚C for 10 min for 2 cycles with a DNA Themal Cycler (TAKARA BIO INC., Shiga, Japan). Gold was required per gel plate. Prestain method. Appropriate dilutions of SYBR Gold were Electrophoresis and detection mixed with 300 ng samples of φX174/HaeIII digest (Marker 4), A submarine system (ATTO, Tokyo, φX174/HincII digest (Marker 5), and pUC19/MspI digest Japan) was used. (Marker and MVR-PCR products) (Marker 11) in a 9 µL volume. A gel loading buffer (1 µL) was were electrophoresed on 3% Agarose 21 gel using TBE buffer added to each sample. A mixture of Marker and SYBR Gold as an electrolyte solution. As a staining reagent, SYBR Gold (10 µL) was applied to 3% agarose 21 gel and separated. For was used, and the DNA bands were detected with a prestaining the PCR products, SYBR Gold was added directly fluoroimageanalyzer FLA-2000 (FUJIFILM, Tokyo, Japan) set to 9 µL of PCR products to make a final dilution of 1:10000. at 473 nm for excitation, and above 520 nm for emission using a Gel loading buffer (1 µL) was added to each sample. The 520 nm cut filter. mixtures (10 µL) of PCR products and SYBR Gold were applied to 3% agarose 21 gel and separated. In this method, Comparison of three different staining methods only 0.001 µL of the stock solution of SYBR Gold was required For the staining of DNAs, three different methods were per lane. examined, as follows. Poststain method. The gel after electrophoresis was immersed in 100 mL of a 1:10000 dilution of SYBR Gold for 40 min at Results and Discussion room temperature. In this method, 10 µL of the stock solution of SYBR Gold was required per run. Comparison of staining methods In-gel stain method. DNAs were separated through gel Figure 1 shows gel electropherograms of Markers 4, 5, and 11 containing SYBR Gold. The SYBR Gold was diluted 100-fold, stained by three staining methods (the prestain method, the in- and a 250-µL aliquot of the solution was added to 25 mL of a gel stain method, and the poststain method) using a 10000-fold 3% melted agarose solution after cooling just before diluted SYBR Gold solution (prestain method and poststain coagulation, thoroughly mixed and immediately poured into the method) or a precasting gel containing the same concentration mold. In this method, 2.5 µL of the stock solution of SYBR of SYBR Gold (in-gel stain method). Significant differences ANALYTICAL SCIENCES JUNE 2005, VOL. 21 621

Fig. 2 Separation of Marker 5 prestained with varying concentrations of SYBR Gold in TBE. Marker 5 (300 ng) was mixed with appropriate dilutions of SYBR Gold. Electrophoresis mode, constant voltage (60 V); gel, 3% Agarose 21; SYBR Gold concentration, 20000- (lane 1), 10000- (lane 2), 5000- (lane 3), 1000- (lane 4), and 500-fold dilutions (lane 5). Fig. 3 Comparison of relative mobility of prestained and poststained Marker 4. DNAs loaded on lane 1 was mixed with 10000-fold of SYBR Gold dilutions before electrophoresis and DNAs were observed in their band intensities. The results indicated loaded on lane 2 was stained with 10000-fold of SYBR Gold that the sensitivity of the in-gel stain method was comparable to dilutions after electrophoresis. Electrophoresis mode, constant that of the poststain method, and also that the in-gel stain voltage (60 V); gel, 3% Agarose 21; buffer, TBE buffer; SYBR Gold method showed a higher separation efficiency compared to the concentration, 10000-fold dilution. The total amount of Marker latter. For example, the DNA bands of 281 bp and 271 bp loaded per lane was 300 ng. (asterisk) in Marker 4 were not separated by the poststain method, whereas these bands were clearly separated by the in- gel stain method. On the other hand, the prestain method showed lower band of ds DNA-dye complexes is known to depend on the dye:DNA intensities. For example, regarding the detection of DNA bands bp ratio5–7 and the concentration ratio of dye to DNA.3–6 To + in Marker 5, 79 bp (about 3 ng/band) was hardly detected, overcome this problem, the use of the Taps-NPe4 buffer system though 162 bp (about 8 ng/band) was detectable. However, the was found to be highly effective.13,14 However, the method + following points are emphasized: (1) the prestain method using the Taps-NPe4 buffer had a high cost, because of the requires only trace amounts of the costly staining dye, and (2) it necessity to use an expensive tetrapentylammonium hydroxide omits the time-consuming process usualy required by 40-min solution. We thus examined the prestaining conditions incubation in the poststain method. Furthermore, SYBR Gold is favorable to a practical application without such a buffer less toxic than ethidium bromide. In particular, the procedure of system. A 10000-fold diluted SYBR Gold was selected as a the prestain method is safer than the other two methods because staining reagent because more than 5000-fold dilution of the it requires only trace amounts of dye. The prestain method reagent tended to lower the migration distortion, though the enabled one to detect bands with as little as 0.003 µL (0.001 band intensities increased with increasing dye concentration µL/lane) of the stock dye solution corresponding to a (Fig. 2). thousandth part of the poststain method, which required a 10-µL Figure 3 shows electropherograms of Marker 4 (300 ng) solution per run with 8 × 10 cm gel. prestained and poststained with 10000-fold dilution SYBR Gold In view of the separation efficiencies, although the in-gel stain in TBE buffer. Their separation profiles seem to be essentially method shows highly efficient separation with high identical. This estimation was further verified as follows. The performance to cost, it is difficult to preserve prestained gel for relative mobilities of the DNA bands observed in both a long time. electropherograms were almost equal in all DNA bands, as Therefore, the prestain method was preferentially studied, as summarized in Fig. 3. Although Tuma et al.1 reported that the described below. detection sensitivity of SYBR Gold (using a SYBR Green/Gold For practical use of the prestain method, there is a problem to photographic filter and 300-nm UV transillumination) for 120 be solved. Figure 2 shows the effect of the SYBR Gold ng samples of HindIII-digested λdsDNA was inadequate at the concentration on the migration profiles of Marker 5. The concentration of dye that did not interfere with the mobility prestain was carried out by the addition of varying (1:29000-fold and greater dilutions), we found based on the concentrations of SYBR Gold (500 – 20000-fold dilutions) to a above finding that a 10000-fold diluted solution of dye fixed amount of Marker 5 (300 ng). The mobility of prestained permitted the detection of DNA without any interference with Marker 5 was decreased with increasing concentration of SYBR the band mobility. An acceptable DNA concentration range in Gold remarkably below its 1000-fold dilution, probably due to which precise mobility was kept was examined using MVR- decreasing the net negative charge of the DNA with increasing PCR products, as described below. cationic dye intercalation. It has been reported that the prestain A SYBR Gold supplier recommended the use of poststain, method is frequently affected by disruptive effects on the beacuse the band deformation occurs upon electrophoresis, migration of the nucleic acids when electrophoresed in the usual leading to a lower resolution in prestaining and in-gel staining. buffer systems, such as TAE or TBE buffers, and the mobility However, at least under the condition described here, these 622 ANALYTICAL SCIENCES JUNE 2005, VOL. 21

Table 1 Sizes of DNA in the MVR-PCR products determined by the standard curve

bp a-type t-type Actual size Calculated size Error, %a Calculated size Error, %a

293 295.32 0.79 —b 322 323.97 0.61 329.36 2.29 351 352.19 0.34 — 380 383.80 1.00 — 409 419.33 2.53 418.26 2.26 438 441.61 0.83 471.74 1.01 467 —b — 496 — 501.91 1.19 525 —— Fig. 4 The migration patterns of MVR-PCR products using the 554 — 556.01 0.36 SYBR Gold prestain method in TBE. Electrophoresis mode, constant 612 — 614.48 0.41 voltage (60 V); gel, 3% Agarose 21; buffer, TBE buffer; samples, 641 — 642.02 0.16 MVR-PCR products (a-type and t-type). Lane M contains 300 ng of 728 — 712.83 –2.08 Marker 5, lane a contains approximately 124 ng of a-type PCR products and lane t contains 98 ng of t-type PCR products. a. Error was calculated as follows: Error = 100 × (calculated size – actual size)/actual size. b. Not detected.

These results clearly demonstrate the utility of the prestain method described here because of its satisfactory features regarding detectability, resolution and size discriminability for dsDNA. Also, these results were accomplished by excitation at 473 nm. To ensure the utility of the prestain method, three staining methods were again tested for the t-type of MVR-PCR products (Fig. 6). The loaded amounts of PCR products not determined, but assumed to be tens–hundreds of ng with increasing order: lane 1 < lane 2 < lane 3. The intensities of bands in the Fig. 5 A plot of the migration distance against 1/log(bp) of Marker poststain method and in the gel stain method were dependent on 5. The migration distances of Marker 5 depicted in Fig. 4 were used. the amount of PCR product. In the poststain method, DNA bands could hardly be detected in lane 1, to which the smallest amount of PCR products was loaded, while a higher background methods have been proved to be usable with several advantages and band broadening were observed in lane 3. In contrast, all rather than the poststain method. DNA bands were clearly detected without band broadening in the prestain method and in the gel stain method. In addition, in Application to MVR-PCR products the prestain method, relatively similar band intensities were The prestain method was applied to the analysis of MVR-PCR obtained regardless of the sample loading amounts. This is a products. Figure 4 shows an analytical result of MVR-PCR unique and desirable property for the qualitative detection of products resulting from the extract of a hair root (the amounts of bands on electropherograms. DNA analyzed were 98 – 124 ng). Excellent separations were The prestain method is concluded to be applicable to establish achieved and 9 – 10 rungs were detected in a-type and t-type a simplified MVR-PCR procedure for individual identification ladders, respectively, which seems to be sufficient to identify an using a hair root, which would be expected to contribute to individual. A plot of the migration distance against 1/log(bp) forensic science. provided a good standard curve (y = 616.93x – 190.24, r2 = 0.9976) (Fig. 5). As shown in Table 1, the sizes of DNA in the MVR-PCR products determined by the standard curve agreed References closely with those of the theoretical values. This proved that the DNA (98 – 124 ng), prestained with a 10000-fold diluted SYBR 1. R. S. Tuma, M. P. Beaudet, X. Jin, L. J. Jones, C. Cheung, Gold solution, migrated without any mobility distortion. S. Yue, and V. L. Singer, Anal. Biochem., 1999, 268, 278. Consequently, for at least the range of about 100 ng to 300 ng of 2. WAKO BIOWINDOW, 1999, 19, 4. the DNA mixture, the prestain method could be used for their 3. H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. precise detection. The range covered most of the cases in our Haugland, R. A. Mathies, and A. N. Glazer, Nucleic Acids MVR-PCR experiments, because the DNA concentration was Res., 1992, 20, 2803. usually about 500 – 600 ng/PCR mixture, corresponding to 4. S. C. Benson, R. A. Mathies, and A. N. Glazer, Nucleic about 100 ng in a 9-µL aliquot used for the application to Acids Res., 1993, 21, 5720. agarose gel. The detectable coverage of the prestain method is 5. Z. Zeng, S. C. Benson, and A. N. Glazer, Anal. Biochem., quite convenient in practical use, because the PCR mixture can 1995, 231, 256. be directly applied to agarose gel without prior concentration or 6. H. S. Rye, S. Yue, M. A. Quesada, R. P. Haugland, R. A. dilution. Mathies, and A. N. Glazer, Methods Enzymol., 1993, 217, ANALYTICAL SCIENCES JUNE 2005, VOL. 21 623

Fig. 6 Comparison of the migration patterns of MVR-PCR products (t-type) on DNA extracted from plucked hair roots originating from three individuals using the three staining methods: the prestain, the in-gel stain, and the poststain methods. Electrophoresis mode, constant voltage (60 V); gel, 3% Agarose 21; buffer, TBE buffer.

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