Appl Biochem Biotechnol (2015) 175:400–409 DOI 10.1007/s12010-014-1282-7
Agarose Gel Purification of PCR Products for Denaturing Gradient Gel Electrophoresis Results in GC-Clamp Deletion Guowei Sun & Jinzhou Xiao & Man Lu & Hongming Wang & Xiaobing Chen & Yongxin Yu & Yingjie Pan & Yongjie Wang
Received: 5 May 2014 / Accepted: 2 October 2014 / Published online: 10 October 2014 # Springer Science+Business Media New York 2014
Abstract The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35±0.19 °C) was less than that of PPFS (83.81±0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples. Keywords DGGE . GC-clamp . Deletion . Melt curve . Sequencing
G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang (*) College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China e-mail: [email protected] G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage & Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China
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Introduction The denaturing gradient gel electrophoresis (DGGE) technique was invented by Fischer and Lerman [1] and is able to separate DNA fragments of the same length but different sequences. DGGE analysis of PCR-amplified 16S ribosomal RNA (rRNA) gene fragments has long been used for revealing the genetic and population diversity of microflora in various environments [2], such as sediments [3–5], soil [6, 7], hot springs [8, 9], freshwater [10, 11], seawater [12–14], and animal digestive tracts [15–18]. A GC-clamp, comprising of guanines (G) and cytosines (C) ranging from 30 to 50 bases [19, 20], is attached to the 5′-end of one PCR primer by chemosynthesis and introduced into one end of the amplified DNA fragments [21–23]. The GC-clamp acts as a high melting domain for preventing complete dissociation of double-stranded DNA molecules into single strands in polyacrylamide gels containing a linear gradient of DNA denaturants (urea and formamide). In terms of DNA fragments ≤500 bp, the detection rate of sequence variants can increase from 50 to nearly 100 % with the attachment of a GC-clamp [19, 21]. Thus, the GCclamp is necessary for the DGGE technique. In this study, an unusual phenomenon, which is the number of DGGE bands was significantly reduced or even disappeared, was observed when the DGGE PCR products were extracted and purified from agarose gels. We figure out that it results from the deletion of GCclamp of PCR products.
Materials and Methods Sample Collection and DNA Extraction The seawater samples were collected from East China Sea, and the community DNA was extracted using QIAamp DNA Stool Mini Kit (QIAGEN, Dusseldorf, Germany) after the seawater was filtered through a 0.22-μm pore-sized filter (Millipore, Massachusetts, USA). The crab samples were collected from an aquafarm, Shanghai, China, and the community DNA of crab gut microbiota (CGM) was extracted from crab guts using DNeasy Blood & Tissue Kit (QIAGEN, Dusseldorf, Germany). Genomic DNA of E. coli DH5α was extracted using TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China). All of extracted DNA was stored at −20 °C until processing. PCR Amplification and DGGE Analysis A nested PCR approach was applied to archaeal 16S rRNA gene amplification of seawater samples using the primer sets 21F-958R and Parch519F-Arch915R (see Table 1). The V3 regions of CGM 16S rRNA gene were amplified using the bacterial V3 universal primer pair with a 40-base GCclamp (341F-518R, see Table 1). PCR amplification was performed in a total volume of 50-μl reaction containing 25 μl 2× PCR MasterMix (TIANGEN, Beijing, China), 0.4 μM of each primer, 22 μl ddH2O, and 1 μl of purified DNA. The details of the primers and the PCR conditions are shown in Table 1. Subsequently, 5 μl of each PCR product was electrophoresed at 120 V for 30 min in 2 % (w/v) agarose gel and 1× Tris-acetate EDTA buffer. PCR products were visualized and photographed by using a GEL Imaging System (Bio-Rad Laboratories, CA, USA). After electrophoresis, the gel slice containing the target DNA was excised, and the target DNA fragments were extracted and purified using the Agarose Gel Extraction Kit according to the manufacturer’s instructions (TIANGEN, Beijing, China) (referred to as the products
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Table 1 Primers and PCR conditions used in this study Primer pair Temperature cycling
Primer sequences
References
21F-958R
After an initial denaturation at 95 °C for 5 min; 35 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 90 s; a final extension of 72 °C for 10 min.
21F: 5′-TTCCGGTTGA Vissers et al. and TCCYGCCGGA-3′ DeLong [10, 24]
Parch519F- After an initial denaturation at 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 57 °C for 40 s, Arc72 °C for 40 s; a final extension of 72 °C for h915R 10 min.
Parch519F: 5′-CAGC Vissers et al. and Coolen et al. [10, CGCCGCGGTA 25] A-3′
958R: 5′-YCCGGCGT TGAMTCCAATT-3′
Arch915R: 5′-GTGC TCCCCCGCCAAT TCCT-3′a
341F: 5′-CCTACGGG Yu and Morrison 341F-518R After an initial denaturation at 94 °C for 5 min, AGGCAGCAG-3′b 20 cycles of touchdown PCR were performed and Mühling (denaturation at 94 °C for 30 s, annealing for et al. [26, 27] 518R: 5′-ATTACCGC 30 s with an −0.5 °C/cycle from 65 °C to GGCTGCTGG-3′ 55 °C, and extension at 72 °C for 1 min), followed by 15 cycles of regular PCR (94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min), and a final extension step at 72 °C for 7 min. a
The 40-nucleotide GC-clamps of 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3′ [2] ] were attached to the 5′-end of primer Arch915R
b
The 40-nucleotide GC-clamps of 5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′ [2] were attached to the 5′-end of primer 341F
purified from gel (PPFGs)). In parallel, the PCR products were also purified directly using the Universal DNA Purification Kit (TIANGEN, Beijing, China) (referred to as products purified from reaction solution (PPFSs)). As for DGGE, 10 μl of each unpurified PCR product (referred to as UPP), PPFS, and PPFG was loaded on a 8 % (w/v) polyacrylamide gel with a 40∼60 % linear gradient of DNA denaturant agents (urea and formamide). The DGGE gel was electrophoresed in 1× Trisacetate EDTA buffer at 60 °C and a constant voltage of 100 V for 17.5 h (DCode system, BioRad Laboratories, CA, USA). DGGE of V3 regions was similar to the conditions described above except for a constant voltage of 60 V for 16 h. The DGGE gel was stained in 1× SYBR Green I (Invitrogen, CA, USA) for 20 min and visualized using a GEL Imaging System (BioRad Laboratories, CA, USA). Cloning of Purified PCR Products The purified PCR products of both PPFS and PPFG were ligated into pGM-T vector by using two methods—TA cloning and blunt-ended cloning—according to the manufacturer’s instructions (TIANGEN, Beijing, China). The recombinant plasmid was transferred into E. coli TOP10 competent cells (TIANGEN, Beijing, China) following the manufacturer’s instructions. Inserts of the expected size (approximately 230 bp) were confirmed by colony PCR. Melt Curve and Direct Sequencing Analysis The V3 regions of E. coli 16S rRNA gene were amplified and purified according to the procedure described in “PCR Amplification and DGGE Analysis” section. Then, both PPFS
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and PPFG were subjected to melt curve analysis using a 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). The reaction was performed in a total volume of 20 μl containing 10 μl 2× FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland), 8 μl ddH2O, and 2 μl of PPFS or PPFG, with a plate read every 0.5 °C from 60 to 95 °C. In parallel, both PPFS and PPFG of V3 regions of E. coli 16S rRNA gene were sequenced directly using the chain termination method and primer 518R by (Sangon, Shanghai, China). In order to reduce sequencing errors for the GC-rich end of the DNA fragments, 100× sequencings were performed for both samples. Ultraviolet Exposure Experiment The UPP of V3 region of CGM 16S rRNA gene was divided into seven aliquots (20 μl/ aliquot). Subsequently, they were exposed to ultraviolet irradiation for 10, 30, 60, 90, 120, 150, and 180 s, respectively, by using the GEL Imaging System (Bio-Rad Laboratories, USA) and subjected to DGGE analysis.
Results PCR Amplification and DGGE Analysis of Marine Archaeal 16S rRNA Gene After PCR amplification by using primers Parch519F/Arch915R with GC-clamp and electrophoresis, some strong nonspecific bands were observed on the agarose gels for the marine archaeal samples (Fig. 1a) when the purified PCR products, obtained by using 21F-958R without the GC-clamp, were used as the templates. Although the nested PCR was optimized systematically, e.g., concentration of DNA template and primers, annealing temperature, and cycle numbers, in order to get rid of these nonspecific amplicons, it did not work (data not shown). Subsequently, the gel slice containing the target DNA was excised and subject to extraction and purification of the target DNA fragments. Accordingly, the nonspecific DNA fragments present in the UPP and PPFS were absent in the PPFG (Fig. 1a).
Fig. 1 Agarose gel images of PCR-amplified 16S rRNA gene fragments obtained by using different templates and primer pairs. a Marine archaeal genomic DNA and archaeal nested PCR primer pair Parch519F-Arch915R. b Crab gut microbiota genomic DNA and bacterial V3 region universal primer pair 341F-518R. c E. coli genomic DNA and bacterial V3 region universal primer pair 341F-518R. N is the negative control
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As for DGGE, in contrast to almost identical band patterns shared by both the UPP and PPFS, surprisingly, the PPFG was barely observable on the DGGE gel (Fig. 2a). DGGE Analysis of CGM V3 Regions In order to understand whether the reduction of DGGE bands was a sample-specific or universal phenomenon, the PCR products of CGM V3 regions (Fig. 1b) were analyzed using the identical protocols. Interestingly, like the marine archaeal samples, the PPFG of the CGM sample contained fewer fragments compared with the UPP and PPFS (Fig. 2b). To exclude the potential influences of the gel extraction kit and staining dye, the TIANGEN Gel Extraction Kit and ethidium bromide were replaced with the QIAGEN Gel Extraction Kit and SYBR Green I, respectively. The DGGE results (Fig. 2b) were consistent with those using TIANGEN Kit and ethidium bromide staining. In addition, few distinct bands appeared on the DGGE gels of the PPFG (Fig. 2b). Consequently, it is reasonable to speculate that the GC-
Fig. 2 Denaturing gradient gel images of PCR-amplified 16S rRNA gene fragments of different templates and primer pairs. a Marine archaeal genomic DNA and archaeal nested PCR primer pair Parch519F-Arch915R. b Crab gut microbiota genomic DNA and bacterial V3 region universal primer pair 341F-518R. PPFG of a and b was extracted and purified by using the TIANGEN Gel Extraction Kit, and PPFGQ of b was done with the QIAGEN Gel Extraction Kit. The amount of the PPFS, PPFG, PPFGQ, and PPFGS used for DGGE analysis is the same. The white arrows indicate distinct bands appearing on the DGGE gels after agarose gel purification. PPFGS is stained with SYBR Green I
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clamp was deleted or modified when the target DNA fragments were extracted and purified from the agarose gel. Cloning of CGM V3 Regions To confirm the GC-clamp deletion of the PPFG, cloning was applied to the target DNA fragments. Only a small amount of positive clones was obtained for the PPFS, but no positive clones were obtained for the PPFG regardless of whether a TA or blunt-ended cloning method was used. The positive controls (DNA fragments without the GC-clamp) were normal. This indicated that the PCR products with the GC-clamp appeared to be difficult for cloning and that the failure of cloning for the PPFG possibly results from GC-clamp deletion. Melt Curve and Direct Sequencing Analysis of E. coli V3 Regions To further confirm the GC-clamp deletion, the V3 regions of E. coli 16S rRNA gene were amplified (Fig. 1c), and both the PPFS and PPFG were subjected to melt curve and direct sequencing analyses. As a result, the Tm value of the PPFG (82.35±0.19 °C) was less than that of the PPFS (83.81±0.11 °C; Fig. 3). It is generally accepted that there is a significant positive correlation between the Tm value and the G + C content of DNA sequences, and the variations of melt curves represent different DNA sequences. However, since both the PPFS and PPFG have identical sequences of 16S rRNA gene, the lower Tm value observed for the PPFG should result from GC-clamp deletion or modification. To confirm this speculation, direct sequencing was performed for the products. As shown in Fig. 4, the number of shorter GCclamps was significant higher in the PPFG than in the PPFS. Besides, the remaining sequences
Fig. 3 Melt curve analysis of both the PPFS and PPFG of E. coli V3 regions. The quantity of both the PPFS and PPFG used for analysis is the same. Mean values and standard deviations were obtained from triplicate experiments
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Fig. 4 Sequencing analysis of both the PPFS and PPFG of E. coli V3 regions. One hundred times of sequencing were performed for both samples to reduce sequencing errors on the GC-clamp. The numbers on the right of each row show the relative abundance of sequences with different length of the GC-clamp
were identical for both the PPFG and PPFS (data not shown). Accordingly, melt curve and direct sequencing analysis suggested that the GC-clamp was randomly deleted during the target DNA fragments extracted from the agarose gel. The Effect of Ultraviolet on GC-Clamp Deletion In order to give insight into the potential causers in charge of GC-clamp deletion, the ultraviolet exposure experiment was performed. As shown in Fig. 5, no obvious differences were observed on DGGE profiles within 60-s ultraviolet exposure. Given that time for excising the gel slice in this study is less than 60 s (approximately 30∼45 s), the ultraviolet was not responsible for the deletion of the GC-clamp.
Discussion In the study of the diversity of the archaeal species composition in East China Sea using the primer pairs PARCH340F-PARCH915R [28] or Parch519F-Arch915R [25], it was observed that a direct PCR-DGGE approach was not suitable for the seawater samples because the primers were not specific enough for the archaeal populations (Sun and Wang, unpublished data). To solve this problem, a nested PCR approach was applied to DGGE analysis. However, obvious nonspecific PCR products occurred. Accordingly, agarose gel purification of the PCR products was performed, and the nonspecific DNA fragments present in the UPP and PPFS were absent in the PPFG after purification (Fig. 1a). This indicates that agarose gel purification can remove most, if not all, nonspecific DNA fragments. As for DGGE, in contrast to almost identical band patterns shared by both the UPP and PPFS, surprisingly, the PPFG was barely observable on the DGGE gel. Given that the PPFG was observable on the agarose gel and was quantified spectrophotometrically prior to DGGE (data not shown), the lack of bands unlikely resulted from few DNA molecules in the PPFG. In
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Fig. 5 The effect of ultraviolet exposure on the GC-clamp. The quantity of PCR products used for DGGE analysis is the same
addition, the changing of DGGE profiles was observed in both the marine archaea and the CGM samples, which exclude the possibility of a sample-specific phenomenon. The changing of DGGE profiles was also present when the TIANGEN Gel Extraction Kit and ethidium bromide staining were replaced with the QIAGEN Gel Extraction Kit and SYBR Green I staining, respectively. Accordingly, the potential influences of the gel extraction kit and staining dyes were also excluded. Consequently, it is reasonable to speculate that the GCclamp was deleted or modified when the target DNA fragments were extracted and purified from the agarose gel. We found that the PCR products with GC-clamp can be cloned as observed in [29], but the cloning efficiency is very low for the PPFS. As for the PPFG, the attempt for cloning is unsuccessful. It suggests that the GC-clamp is indeed deleted, which results in the failure of cloning. The melt curve analysis of both the PPFS and PPFG of E. coli V3 regions indicates that the Tm value of the PPFG (82.35±0.19 °C) is less than that of the PPFS (83.81±0.11 °C). Since both the PPFS and PPFG have identical sequence of 16S rRNA gene, the lower Tm value observed for the PPFG should result from GC-clamp deletion. The sequencing results indicate that the number of shorter GC-clamps is significantly higher in the PPFG than in the PPFS. Therefore, we deduce that the GC-clamp is randomly deleted during the target DNA fragments extracted from the agarose gel, which, consequently, led to (i) a significant reduction in the number of DGGE bands (Fig. 2b), or even their disappearance (Fig. 2a), and (ii) some distinct bands appearing on the DGGE gels (Fig. 2b). The results are consistent with the previous study showing that variable GC-clamps can affect the DGGE profiles [29]. Finally, UV light was not responsible for the GC-clamp deletion (Fig. 5). Some unknown reagents in the gel-dissolving buffer supplied with the commercial kits may react with and change the GC-clamps.
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Conclusions Taken together, the results presented here demonstrate that the GC-clamp is deleted following a random and partial mode when the DGGE PCR products are extracted and purified from agarose gels. Accordingly, the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples and should be avoided in similar studies. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 41376135), Doctoral Fund of Ministry of Education of China (20133104110006), Innovation Program of Shanghai Municipal Education Commission (14ZZ144), China, and Construction Program of Shanghai Committee of Science and Technology (11DZ2280300), China.
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24. DeLong, E. F. (1992). Proceedings of the National Academy of Sciences of the United States of America, 89, 5685–5689. 25. Coolen, M. J. L., Hopmans, E. C., Rijpstra, W. I. C., Muyzer, G., Schouten, S., Volkman, J. K., & Damste, J. S. S. (2004). Organic Geochemistry, 35, 1151–1167. 26. Yu, Z., & Morrison, M. (2004). Applied and Environmental Microbiology, 70, 4800–4806. 27. Mühling, M., Woolven-Allen, J., Murrell, J. C., & Joint, I. (2008). The ISME Journal, 2, 379–92. 28. Ovreås, L., Forney, L., Daae, F. L., & Torsvik, V. (1997). Applied and Environmental Microbiology, 63, 3367–3373. 29. Rettedal, E. A., Clay, S., & Brözel, V. S. (2010). FEMS Microbiology Letters, 312, 55–62.
Agarose Gel Purification of PCR Products for Denaturing Gradient Gel Electrophoresis Results in GC-Clamp Deletion Guowei Sun & Jinzhou Xiao & Man Lu & Hongming Wang & Xiaobing Chen & Yongxin Yu & Yingjie Pan & Yongjie Wang
Received: 5 May 2014 / Accepted: 2 October 2014 / Published online: 10 October 2014 # Springer Science+Business Media New York 2014
Abstract The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35±0.19 °C) was less than that of PPFS (83.81±0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples. Keywords DGGE . GC-clamp . Deletion . Melt curve . Sequencing
G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang (*) College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China e-mail: [email protected] G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China G. Sun : J. Xiao : M. Lu : H. Wang : X. Chen : Y. Yu : Y. Pan : Y. Wang Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage & Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China
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Introduction The denaturing gradient gel electrophoresis (DGGE) technique was invented by Fischer and Lerman [1] and is able to separate DNA fragments of the same length but different sequences. DGGE analysis of PCR-amplified 16S ribosomal RNA (rRNA) gene fragments has long been used for revealing the genetic and population diversity of microflora in various environments [2], such as sediments [3–5], soil [6, 7], hot springs [8, 9], freshwater [10, 11], seawater [12–14], and animal digestive tracts [15–18]. A GC-clamp, comprising of guanines (G) and cytosines (C) ranging from 30 to 50 bases [19, 20], is attached to the 5′-end of one PCR primer by chemosynthesis and introduced into one end of the amplified DNA fragments [21–23]. The GC-clamp acts as a high melting domain for preventing complete dissociation of double-stranded DNA molecules into single strands in polyacrylamide gels containing a linear gradient of DNA denaturants (urea and formamide). In terms of DNA fragments ≤500 bp, the detection rate of sequence variants can increase from 50 to nearly 100 % with the attachment of a GC-clamp [19, 21]. Thus, the GCclamp is necessary for the DGGE technique. In this study, an unusual phenomenon, which is the number of DGGE bands was significantly reduced or even disappeared, was observed when the DGGE PCR products were extracted and purified from agarose gels. We figure out that it results from the deletion of GCclamp of PCR products.
Materials and Methods Sample Collection and DNA Extraction The seawater samples were collected from East China Sea, and the community DNA was extracted using QIAamp DNA Stool Mini Kit (QIAGEN, Dusseldorf, Germany) after the seawater was filtered through a 0.22-μm pore-sized filter (Millipore, Massachusetts, USA). The crab samples were collected from an aquafarm, Shanghai, China, and the community DNA of crab gut microbiota (CGM) was extracted from crab guts using DNeasy Blood & Tissue Kit (QIAGEN, Dusseldorf, Germany). Genomic DNA of E. coli DH5α was extracted using TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China). All of extracted DNA was stored at −20 °C until processing. PCR Amplification and DGGE Analysis A nested PCR approach was applied to archaeal 16S rRNA gene amplification of seawater samples using the primer sets 21F-958R and Parch519F-Arch915R (see Table 1). The V3 regions of CGM 16S rRNA gene were amplified using the bacterial V3 universal primer pair with a 40-base GCclamp (341F-518R, see Table 1). PCR amplification was performed in a total volume of 50-μl reaction containing 25 μl 2× PCR MasterMix (TIANGEN, Beijing, China), 0.4 μM of each primer, 22 μl ddH2O, and 1 μl of purified DNA. The details of the primers and the PCR conditions are shown in Table 1. Subsequently, 5 μl of each PCR product was electrophoresed at 120 V for 30 min in 2 % (w/v) agarose gel and 1× Tris-acetate EDTA buffer. PCR products were visualized and photographed by using a GEL Imaging System (Bio-Rad Laboratories, CA, USA). After electrophoresis, the gel slice containing the target DNA was excised, and the target DNA fragments were extracted and purified using the Agarose Gel Extraction Kit according to the manufacturer’s instructions (TIANGEN, Beijing, China) (referred to as the products
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Table 1 Primers and PCR conditions used in this study Primer pair Temperature cycling
Primer sequences
References
21F-958R
After an initial denaturation at 95 °C for 5 min; 35 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 90 s; a final extension of 72 °C for 10 min.
21F: 5′-TTCCGGTTGA Vissers et al. and TCCYGCCGGA-3′ DeLong [10, 24]
Parch519F- After an initial denaturation at 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 57 °C for 40 s, Arc72 °C for 40 s; a final extension of 72 °C for h915R 10 min.
Parch519F: 5′-CAGC Vissers et al. and Coolen et al. [10, CGCCGCGGTA 25] A-3′
958R: 5′-YCCGGCGT TGAMTCCAATT-3′
Arch915R: 5′-GTGC TCCCCCGCCAAT TCCT-3′a
341F: 5′-CCTACGGG Yu and Morrison 341F-518R After an initial denaturation at 94 °C for 5 min, AGGCAGCAG-3′b 20 cycles of touchdown PCR were performed and Mühling (denaturation at 94 °C for 30 s, annealing for et al. [26, 27] 518R: 5′-ATTACCGC 30 s with an −0.5 °C/cycle from 65 °C to GGCTGCTGG-3′ 55 °C, and extension at 72 °C for 1 min), followed by 15 cycles of regular PCR (94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min), and a final extension step at 72 °C for 7 min. a
The 40-nucleotide GC-clamps of 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3′ [2] ] were attached to the 5′-end of primer Arch915R
b
The 40-nucleotide GC-clamps of 5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′ [2] were attached to the 5′-end of primer 341F
purified from gel (PPFGs)). In parallel, the PCR products were also purified directly using the Universal DNA Purification Kit (TIANGEN, Beijing, China) (referred to as products purified from reaction solution (PPFSs)). As for DGGE, 10 μl of each unpurified PCR product (referred to as UPP), PPFS, and PPFG was loaded on a 8 % (w/v) polyacrylamide gel with a 40∼60 % linear gradient of DNA denaturant agents (urea and formamide). The DGGE gel was electrophoresed in 1× Trisacetate EDTA buffer at 60 °C and a constant voltage of 100 V for 17.5 h (DCode system, BioRad Laboratories, CA, USA). DGGE of V3 regions was similar to the conditions described above except for a constant voltage of 60 V for 16 h. The DGGE gel was stained in 1× SYBR Green I (Invitrogen, CA, USA) for 20 min and visualized using a GEL Imaging System (BioRad Laboratories, CA, USA). Cloning of Purified PCR Products The purified PCR products of both PPFS and PPFG were ligated into pGM-T vector by using two methods—TA cloning and blunt-ended cloning—according to the manufacturer’s instructions (TIANGEN, Beijing, China). The recombinant plasmid was transferred into E. coli TOP10 competent cells (TIANGEN, Beijing, China) following the manufacturer’s instructions. Inserts of the expected size (approximately 230 bp) were confirmed by colony PCR. Melt Curve and Direct Sequencing Analysis The V3 regions of E. coli 16S rRNA gene were amplified and purified according to the procedure described in “PCR Amplification and DGGE Analysis” section. Then, both PPFS
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and PPFG were subjected to melt curve analysis using a 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). The reaction was performed in a total volume of 20 μl containing 10 μl 2× FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland), 8 μl ddH2O, and 2 μl of PPFS or PPFG, with a plate read every 0.5 °C from 60 to 95 °C. In parallel, both PPFS and PPFG of V3 regions of E. coli 16S rRNA gene were sequenced directly using the chain termination method and primer 518R by (Sangon, Shanghai, China). In order to reduce sequencing errors for the GC-rich end of the DNA fragments, 100× sequencings were performed for both samples. Ultraviolet Exposure Experiment The UPP of V3 region of CGM 16S rRNA gene was divided into seven aliquots (20 μl/ aliquot). Subsequently, they were exposed to ultraviolet irradiation for 10, 30, 60, 90, 120, 150, and 180 s, respectively, by using the GEL Imaging System (Bio-Rad Laboratories, USA) and subjected to DGGE analysis.
Results PCR Amplification and DGGE Analysis of Marine Archaeal 16S rRNA Gene After PCR amplification by using primers Parch519F/Arch915R with GC-clamp and electrophoresis, some strong nonspecific bands were observed on the agarose gels for the marine archaeal samples (Fig. 1a) when the purified PCR products, obtained by using 21F-958R without the GC-clamp, were used as the templates. Although the nested PCR was optimized systematically, e.g., concentration of DNA template and primers, annealing temperature, and cycle numbers, in order to get rid of these nonspecific amplicons, it did not work (data not shown). Subsequently, the gel slice containing the target DNA was excised and subject to extraction and purification of the target DNA fragments. Accordingly, the nonspecific DNA fragments present in the UPP and PPFS were absent in the PPFG (Fig. 1a).
Fig. 1 Agarose gel images of PCR-amplified 16S rRNA gene fragments obtained by using different templates and primer pairs. a Marine archaeal genomic DNA and archaeal nested PCR primer pair Parch519F-Arch915R. b Crab gut microbiota genomic DNA and bacterial V3 region universal primer pair 341F-518R. c E. coli genomic DNA and bacterial V3 region universal primer pair 341F-518R. N is the negative control
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As for DGGE, in contrast to almost identical band patterns shared by both the UPP and PPFS, surprisingly, the PPFG was barely observable on the DGGE gel (Fig. 2a). DGGE Analysis of CGM V3 Regions In order to understand whether the reduction of DGGE bands was a sample-specific or universal phenomenon, the PCR products of CGM V3 regions (Fig. 1b) were analyzed using the identical protocols. Interestingly, like the marine archaeal samples, the PPFG of the CGM sample contained fewer fragments compared with the UPP and PPFS (Fig. 2b). To exclude the potential influences of the gel extraction kit and staining dye, the TIANGEN Gel Extraction Kit and ethidium bromide were replaced with the QIAGEN Gel Extraction Kit and SYBR Green I, respectively. The DGGE results (Fig. 2b) were consistent with those using TIANGEN Kit and ethidium bromide staining. In addition, few distinct bands appeared on the DGGE gels of the PPFG (Fig. 2b). Consequently, it is reasonable to speculate that the GC-
Fig. 2 Denaturing gradient gel images of PCR-amplified 16S rRNA gene fragments of different templates and primer pairs. a Marine archaeal genomic DNA and archaeal nested PCR primer pair Parch519F-Arch915R. b Crab gut microbiota genomic DNA and bacterial V3 region universal primer pair 341F-518R. PPFG of a and b was extracted and purified by using the TIANGEN Gel Extraction Kit, and PPFGQ of b was done with the QIAGEN Gel Extraction Kit. The amount of the PPFS, PPFG, PPFGQ, and PPFGS used for DGGE analysis is the same. The white arrows indicate distinct bands appearing on the DGGE gels after agarose gel purification. PPFGS is stained with SYBR Green I
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clamp was deleted or modified when the target DNA fragments were extracted and purified from the agarose gel. Cloning of CGM V3 Regions To confirm the GC-clamp deletion of the PPFG, cloning was applied to the target DNA fragments. Only a small amount of positive clones was obtained for the PPFS, but no positive clones were obtained for the PPFG regardless of whether a TA or blunt-ended cloning method was used. The positive controls (DNA fragments without the GC-clamp) were normal. This indicated that the PCR products with the GC-clamp appeared to be difficult for cloning and that the failure of cloning for the PPFG possibly results from GC-clamp deletion. Melt Curve and Direct Sequencing Analysis of E. coli V3 Regions To further confirm the GC-clamp deletion, the V3 regions of E. coli 16S rRNA gene were amplified (Fig. 1c), and both the PPFS and PPFG were subjected to melt curve and direct sequencing analyses. As a result, the Tm value of the PPFG (82.35±0.19 °C) was less than that of the PPFS (83.81±0.11 °C; Fig. 3). It is generally accepted that there is a significant positive correlation between the Tm value and the G + C content of DNA sequences, and the variations of melt curves represent different DNA sequences. However, since both the PPFS and PPFG have identical sequences of 16S rRNA gene, the lower Tm value observed for the PPFG should result from GC-clamp deletion or modification. To confirm this speculation, direct sequencing was performed for the products. As shown in Fig. 4, the number of shorter GCclamps was significant higher in the PPFG than in the PPFS. Besides, the remaining sequences
Fig. 3 Melt curve analysis of both the PPFS and PPFG of E. coli V3 regions. The quantity of both the PPFS and PPFG used for analysis is the same. Mean values and standard deviations were obtained from triplicate experiments
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Fig. 4 Sequencing analysis of both the PPFS and PPFG of E. coli V3 regions. One hundred times of sequencing were performed for both samples to reduce sequencing errors on the GC-clamp. The numbers on the right of each row show the relative abundance of sequences with different length of the GC-clamp
were identical for both the PPFG and PPFS (data not shown). Accordingly, melt curve and direct sequencing analysis suggested that the GC-clamp was randomly deleted during the target DNA fragments extracted from the agarose gel. The Effect of Ultraviolet on GC-Clamp Deletion In order to give insight into the potential causers in charge of GC-clamp deletion, the ultraviolet exposure experiment was performed. As shown in Fig. 5, no obvious differences were observed on DGGE profiles within 60-s ultraviolet exposure. Given that time for excising the gel slice in this study is less than 60 s (approximately 30∼45 s), the ultraviolet was not responsible for the deletion of the GC-clamp.
Discussion In the study of the diversity of the archaeal species composition in East China Sea using the primer pairs PARCH340F-PARCH915R [28] or Parch519F-Arch915R [25], it was observed that a direct PCR-DGGE approach was not suitable for the seawater samples because the primers were not specific enough for the archaeal populations (Sun and Wang, unpublished data). To solve this problem, a nested PCR approach was applied to DGGE analysis. However, obvious nonspecific PCR products occurred. Accordingly, agarose gel purification of the PCR products was performed, and the nonspecific DNA fragments present in the UPP and PPFS were absent in the PPFG after purification (Fig. 1a). This indicates that agarose gel purification can remove most, if not all, nonspecific DNA fragments. As for DGGE, in contrast to almost identical band patterns shared by both the UPP and PPFS, surprisingly, the PPFG was barely observable on the DGGE gel. Given that the PPFG was observable on the agarose gel and was quantified spectrophotometrically prior to DGGE (data not shown), the lack of bands unlikely resulted from few DNA molecules in the PPFG. In
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Fig. 5 The effect of ultraviolet exposure on the GC-clamp. The quantity of PCR products used for DGGE analysis is the same
addition, the changing of DGGE profiles was observed in both the marine archaea and the CGM samples, which exclude the possibility of a sample-specific phenomenon. The changing of DGGE profiles was also present when the TIANGEN Gel Extraction Kit and ethidium bromide staining were replaced with the QIAGEN Gel Extraction Kit and SYBR Green I staining, respectively. Accordingly, the potential influences of the gel extraction kit and staining dyes were also excluded. Consequently, it is reasonable to speculate that the GCclamp was deleted or modified when the target DNA fragments were extracted and purified from the agarose gel. We found that the PCR products with GC-clamp can be cloned as observed in [29], but the cloning efficiency is very low for the PPFS. As for the PPFG, the attempt for cloning is unsuccessful. It suggests that the GC-clamp is indeed deleted, which results in the failure of cloning. The melt curve analysis of both the PPFS and PPFG of E. coli V3 regions indicates that the Tm value of the PPFG (82.35±0.19 °C) is less than that of the PPFS (83.81±0.11 °C). Since both the PPFS and PPFG have identical sequence of 16S rRNA gene, the lower Tm value observed for the PPFG should result from GC-clamp deletion. The sequencing results indicate that the number of shorter GC-clamps is significantly higher in the PPFG than in the PPFS. Therefore, we deduce that the GC-clamp is randomly deleted during the target DNA fragments extracted from the agarose gel, which, consequently, led to (i) a significant reduction in the number of DGGE bands (Fig. 2b), or even their disappearance (Fig. 2a), and (ii) some distinct bands appearing on the DGGE gels (Fig. 2b). The results are consistent with the previous study showing that variable GC-clamps can affect the DGGE profiles [29]. Finally, UV light was not responsible for the GC-clamp deletion (Fig. 5). Some unknown reagents in the gel-dissolving buffer supplied with the commercial kits may react with and change the GC-clamps.
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Conclusions Taken together, the results presented here demonstrate that the GC-clamp is deleted following a random and partial mode when the DGGE PCR products are extracted and purified from agarose gels. Accordingly, the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples and should be avoided in similar studies. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 41376135), Doctoral Fund of Ministry of Education of China (20133104110006), Innovation Program of Shanghai Municipal Education Commission (14ZZ144), China, and Construction Program of Shanghai Committee of Science and Technology (11DZ2280300), China.
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