- Email: [email protected]
Evidence for chimeric sequences formed during random arbitrarily primed PCR
Journal of Microbiological Methods 54 (2003) 427 – 431 www.elsevier.com/locate/jmicmeth
Note
Evidence for chimeric sequences formed during random arbitrarily primed PCR In Seop Chang a,1, Jimmy D. Ballard a, Lee R. Krumholz a,b,* a b
Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019-0245, USA Institute for Energy and the Environment, University of Oklahoma, Norman, OK 73019-0245, USA
Received 10 December 2002; received in revised form 14 February 2003; accepted 7 March 2003
Abstract Chimeric sequences were observed to occur abundantly (48% of clones) during random arbitrarily primed polymerase chain reaction (RAP-PCR) experiments designed to examine differential expression of genes involved in metal resistance in sulfatereducing bacteria (SRB). Some of the chimeric sequences were composed of sequence from a gene differentially expressed under the imposed conditions and a sequence of the 16S or 23S rRNA gene. The remainder were composed of two rRNA sequences. Experiments using PCR and genomic sequence analysis showed that the chimeric sequences were not due to a genetic mutation (e.g., recombination, transposition). As RAP-PCR has been widely used to identify differentially expressed genes, this observation may aid in our interpretation of RAP-PCR data. D 2003 Elsevier Science B.V. All rights reserved.
In 1992, Liang and Pardee (1992) showed that differential display (DD) PCR was effective for amplification and display of cDNAs derived from mRNA. They designed two different types of synthetic oligonucleotides for this purpose: anchored and arbitrary primers. Anchored primers have complementary sequences to the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. With these primers, RNA and reverse transcriptase, the primed reaction results in synthesis of cDNA. * Corresponding author. Department of Botany and Microbiology, University of Oklahoma, George Lynn Cross Hall, 770 Van Vleet Oval, Norman, OK 73019, USA. Tel.: +1-405-325-0437; fax: +1-405-325-7619. E-mail address: [email protected] (L.R. Krumholz). 1 Present address: Water Environment and Remediation Center, KIST, 39-1, Hawolgok-dong, Sungpook-ku, Seoul 136-791, Korea.
Following the RT reaction, a second arbitrary primer is added to the reaction mixture and double stranded cDNAs are amplified by conventional PCR under low stringency conditions. As prokaryotic mRNA does not have the poly(A) tail, arbitrary primers are typically used for both the RT and PCR steps (McClelland et al., 1997; Rothschild et al., 1997), and this process is termed random arbitrarily primed (RAP) PCR. PCR products are visualized after separation on denaturing polyacrylamide gels, and profiles of cells grown under different conditions are compared. Unique RNAs are then cloned and sequenced. Although these methods have been widely used because of their sensitivity and relative low cost, problems have been reported, which include false positives (Faslage et al., 1997; Nagel et al., 1999) and occasional irreproducibility (Liang and Pardee, 1992;
0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00093-9
428
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
Mou et al., 1994). In addition, even if the amplified product is exactly cut out, the PCR product may be a mixture of products of similar size (Mathieu-Daude et al., 1996). Here we report that a fraction of false positive bands, originally identified as rRNA products, are actually chimeric sequences some of which are composed of rRNA and a differentially expressed gene product (mRNA). Our specific interests are in the response of sulfatereducing bacteria (SRB) to metals at the genetic level. SRB are important microorganisms in a variety of anaerobic ecosystems, including those contaminated with metals (Sani et al., 2001). Several metal bioremediation approaches, in situ (Waybrant et al., 1998) and bioreactor (Chang et al., 2000; White and Gadd, 1996) based, have been proposed, which involved SRB. We have attempted to identify genes expressed in response to metals using random arbitrarily primed polymerase chain reaction (RAP-PCR). Desulfovibrio vulgaris Hildenborough was used as the model strain throughout the study, and Cu(II) was used as a heavy metal. D. vulgaris was cultivated anaerobically under fermentative conditions in a mineral medium containing 0.2% yeast extract, 0.0001% resazurin (redox indicator), vitamins, minerals, and trace metals prepared as previously described (Krumholz and Bryant, 1986). The medium contained 20 mM pyruvate as electron acceptor and was treated with/without 50 AM CuCl2. When cultures were in early log phase, total RNA was isolated as described by Steger et al. (2001). RAP-PCR was then performed as described by Shepard and Gilmore (1999). The sequences of the arbitrary primers were AATCTAGAGCTCTCCTGG (primer 1) and AATCTAGAGCTCCCTCCA (primer 2). Products were labeled with 33P-dCTP and visualized by running on a denaturing polyacrylamide gel (6%) at 1,200 V for 10 h. The gel was dried and exposed to Kodak BioMax MR film as well as a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA USA) for 18 h at room temperature. Fig. 1 shows the cDNA profiles of RAPPCR. The two candidate bands (band #1 and band #2) were excised from the gel, eluted with buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS, precipitated with 100% ethanol, and resuspended in 10 Al sterile water. The cDNA was then reamplified and resolved on an identical gel loaded adjacent to the original RAP-PCR product to ensure that the correct band was reamplified
Fig. 1. cDNA band profiles on denaturing gels after RAP-PCR using primer 2. Lanes 1 – 7: 50 AM CuCl2 treatment and lanes 8 – 13, control (no treatment).
(data not shown). The candidate PCR product was ligated into pCR4-TOPO vector (Invitrogen, Carlsbad, CA, USA) and transformed into chemically competent One Shot TOP10 E. coli (Invitrogen). Plasmid was isolated from 3 ml of liquid cultures of candidate clones grown in LB + ampicillin (100 Ag/ml) using a plasmid isolation kit (Qiaprep Spin Miniprepkit, QIAGEN, Valenica, CA, USA) according to the manufacturer’s directions. After plasmid isolation, the plasmids were cut with EcoR1 (Invitrogen) and resolved on a
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
0.8% (w/v) agarose gel to compare inserts (data not shown). DNA sequencing was carried out at the Oklahoma Medical Research Foundation (Oklahoma City, OK) Core Sequencing Facility. For this study, we cloned 8 bands and picked 10 clones to be sequenced per transformation. Candidate insert sequences were compared to the whole genome of D. vulgaris Hildenborough in TIGR BLAST Search Engine for Unfinished Microbial Genomes (UFMG). Fig. 2 shows an example sequence of band #1 that contains sequences from two different open reading frames but shared an identical DNA sequence of 29 base pairs. These sequences were found within the genomic sequence of D. vulgaris but were separated by about 300 kb of genomic DNA. This type of result occurred in an average of 5 of every 10 clones sequenced and occurred
429
with both of the tested arbitrary primers. Of the 36 chimeric sequences observed, 3 contained chimeras of mRNA and rRNA, and the remainder were of 2 different rRNA sequences. The remaining nonchimeric sequences were all of 16S or 23S rRNA. In order to be certain that the observed sequences did not occur as a result of genetic recombination within the sulfate-reducing bacterium, we designed primer sets (boxed sequences in Fig. 2A) to amplify each of the two genes on a chimeric sequence and amplified chromosomal and plasmid DNA as templates. Chromosomal DNA was isolated using Easy-DNAk kit (Invitrogen) according to the manufacturer’s directions. To initiate PCR experiments, 1 Ag chromosomal DNA or cloned plasmid DNA was added to a 0.65 ml thin-wall PCR tube with 5 Al buffer (10X buffer,
Fig. 2. The chimeric sequence (A) and schematic of TIGR whole genome search results (B) shown in the same order. The sequence was obtained from one of the clones (band #1 in Fig. 1), and a whole genome search was performed using Unfinished Microbial Genome (UFMG) in TIGR (http://tigrblast.tigr.org/ufmg). Specific primer sets (indicated by boxed regions) were designed to reamplify each gene of the chimeric sequence for which results are presented in Fig. 3. The underlined sequence is identical to that present in one of the genes and similar to that present in the second gene. Random primer sequence is not shown.
430
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
Invitrogen), 1 Al dNTP (10 mM), 1.5 Al MgCl2 (50 mM), 1 Al primer mixture (50 mM of each forward and reverse primer), 0.5 Al Taq polymerase (5 U/Al, Invitrogen), and the total volume was adjusted to 50 Al with water. The reaction mixture was incubated at 95 jC for 10 min, and the following parameters were used for an additional 30 cycles of PCR: 94 jC (1 min), 56 jC (1 min), 72 jC (1 min), and a final extension at 72 jC for 10 min. Fig. 3A shows the 1.5% agarose gel electrophoresis (120 V for 30 min) results of band #1. The predicted PCR product size for the two individual open reading frames were observed with both chromosomal
Fig. 3. Electrophoresis results confirming the chimeric sequence in clones. The chimeric sequence obtained from band #1 (A) and band #2 (B). The predicted PCR product size for the two individual open reading frames were observed with both cloned (lanes 1 and 2) and chromosomal (lanes 4 and 5) DNA. The PCR product of the chimeric sequence was observed with the cloned DNA (lane 6) but not with the chromosomal DNA (lane 3).
(lanes 4 and 5) and cloned (lanes 1 and 2) DNA (Fig. 3A). However, when we attempted to amplify the entire chimeric sequence, a product was observed with the cloned DNA (lane 6) but not with chromosomal DNA (lane 3). Other chimeric sequences (band #2) were examined with similar results (Fig. 3). Because only one random primer is used for both first (RT reaction) and second strand synthesis, the cloned sequences are flanked by the same primer sequence. The primer sequence was not observed in the middle of the cloned sequence. However, when chimeric sequences are used to search the whole genome (UFMG) D. vulgaris database, we always observed a region where sequence identity or high homology exists between the two sequences within the chimera. In D. vulgaris, the whole genome has five copies of 23S and 16S rDNA located throughout the genome. As shown in Fig. 2B, the chimeric sequence has a sequence derived from 23S rRNA and another sequence that could be derived from mRNA. This result indicates that even if RAP-PCR products have a high similarity to rRNA sequence after BLAST searches, it is possible that they could contain important information. In fact, we have shown that some of the mRNA sequences within the chimeras are from induced mRNAs (data not shown). The occurrence of chimeric clones during more traditional PCR based cloning experiments is much more rare (Speksnijder et al., 2001) than we have observed using RAP-PCR. The major difference being that with RAP-PCR, RNA is the template and random primers are being used. A common feature of chimeric sequences is the sequence near the middle common to both rRNA and mRNA or to two rRNAs. This region ranged in size from 9 to 64 base pairs in our clones. Typically, the common sequence shows identity to one sequence from the database and similarity to the second sequence. Because of this feature and based on the relative abundance of chimeric clones, we suggest that chimeras likely occur as two cDNAs may form a duplex during the early stage of the process. Random primers bind to only one site on each of the strands and likely amplify the entire duplex. One of the major problems with DD- or RAP-PCR techniques is the high incidence of false positive bands, which turn out to have rRNA sequences (Faslage et al., 1997; Nagel et al., 1999, 2001). This is typically attributed to the relative abundance of rRNA in the total RNA pool. Several research groups
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
have attempted to minimize these false results using random primers specifically designed to avoid rRNA amplification (Faslage et al., 1997; Nagel et al., 1999). However, this method is likely to be of limited use because of the requirement for genomic sequence information. Recently, Nagel et al. (2001) described a screening method using oligonucleotide probes designed to identify and ultimately eliminate clones derived from rRNAs. Colony blots were prepared and those colonies, which did not hybridize to rRNA probes, were used to identify genes in Pseudomonas expressed in response to Cadmium exposure. Although these approaches could be used to reduce the incidence of false positive results, our experiments demonstrated that rRNA containing sequences often contain useful information. As rRNA containing chimeric sequences was observed in mRNA containing clones, it is unwise to eliminate them. Because RAPPCR has been widely used to identify transcripts differentially expressed under altered conditions, this finding could be useful for interpretation of data from DD- and RAP-PCR.
Acknowledgements This research was supported by a grant from the Natural and Accelerated Bioremediation Research (NABIR) program of the Office of Biological and Environmental Research of the U.S. Department of Energy, Office of Science.
References Chang, I.S., Shin, P.K., Kim, B.H., 2000. Biological treatment of acid mine drainage under sulphate-reducing conditions with solid waste materials as substrate. Water Res. 34, 1269 – 1277. Faslage, R., Berceanu, M., Humboldt, Y., Wendt, M., Oberender, H., 1997. Primer design for a prokaryotic differential display RT-PCR. Nucleic Acids Res. 25, 1830 – 1835. Krumholz, L.R., Bryant, M.P., 1986. Eubacterium oxidoreducens
431
sp. nov. requiring H2 or formate to degrade gallate, pyrogallol, phloroglucinol and quercetin. Arch. Microbiol. 144, 8 – 14. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967 – 971. Mathieu-Daude, F., Cheng, R., Welsh, J., McClelland, M., 1996. Screening of differently amplified cDNA products from RNA arbitrarily primer PCR fingerprints using single strand conformation polymorphism (SSCP) gels. Nucleic Acids Res. 24, 1504 – 1507. McClelland, M., Honeycutt, R., Mathieu-Daude, F., Vogt, T., Welsh, J., 1997. Finger printing by arbitrarily primer PCR. Methods Mol. Biol. 85, 13 – 24. Mou, L., Miller, H., Li, J., Wang, E., Chalofour, L., 1994. Improvements to the differential display method for gene analysis. Biochem. Biophys. Res. Commun. 199, 564 – 569. Nagel, A., Fleming, J.T., Sayler, G.S., 1999. Reduction of false positives in prokaryotic mRNA differential display. BioTechniques 26, 641 – 648. Nagel, A.C., Fleming, J.T., Sayler, G.S., Beattie, K.L., 2001. Screening for ribosomal-based false positives following prokaryotes mRNA differential display. BioTechniques 30, 988 – 996. Rothschild, D.B., Brewer, C.S., Bowden, D.W., 1997. DD/APPCR: combination of differential display and arbitrarily primed PCR of oligo (dt) cDNA. Anal. Biochem. 245, 48 – 54. Sani, R.K., Peyton, B.M., Brown, L.T., 2001. Copper-induced inhibition of growth of Desulfovibrio desulfuricans G20: assessment of its toxicity and correlation with those of zinc and lead. Appl. Environ. Microbiol. 67, 4765 – 4772. Shepard, B., Gilmore, M., 1999. Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR. Appl. Environ. Microbiol. 65, 1470 – 1476. Speksnijder, A.G.C.L., Kowalchuk, G.A., De Jong, S., Kline, E., Stephen, J.R., Laanbroek, H.J., 2001. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl. Environ. Microbiol. 67, 469 – 472. Steger, J.L., Vincent, C., Ballard, J.D., Krumholz, L.R., 2001. Desulfovibrio sp. genes involved in the respiration of sulfate during metabolism of hydrogen and lactate. Appl. Environ. Microbiol. 68, 1932 – 1947. Waybrant, K.R., Blowes, D.W., Ptacek, C.J., 1998. Selection of reactive mixtures for use in permeable reactive walls for treatment of mine drainage. Environ. Sci. Technol. 32, 1972 – 1979. White, C., Gadd, G.M., 1996. Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration. Microbiology 142, 2197 – 2205.
Note
Evidence for chimeric sequences formed during random arbitrarily primed PCR In Seop Chang a,1, Jimmy D. Ballard a, Lee R. Krumholz a,b,* a b
Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019-0245, USA Institute for Energy and the Environment, University of Oklahoma, Norman, OK 73019-0245, USA
Received 10 December 2002; received in revised form 14 February 2003; accepted 7 March 2003
Abstract Chimeric sequences were observed to occur abundantly (48% of clones) during random arbitrarily primed polymerase chain reaction (RAP-PCR) experiments designed to examine differential expression of genes involved in metal resistance in sulfatereducing bacteria (SRB). Some of the chimeric sequences were composed of sequence from a gene differentially expressed under the imposed conditions and a sequence of the 16S or 23S rRNA gene. The remainder were composed of two rRNA sequences. Experiments using PCR and genomic sequence analysis showed that the chimeric sequences were not due to a genetic mutation (e.g., recombination, transposition). As RAP-PCR has been widely used to identify differentially expressed genes, this observation may aid in our interpretation of RAP-PCR data. D 2003 Elsevier Science B.V. All rights reserved.
In 1992, Liang and Pardee (1992) showed that differential display (DD) PCR was effective for amplification and display of cDNAs derived from mRNA. They designed two different types of synthetic oligonucleotides for this purpose: anchored and arbitrary primers. Anchored primers have complementary sequences to the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. With these primers, RNA and reverse transcriptase, the primed reaction results in synthesis of cDNA. * Corresponding author. Department of Botany and Microbiology, University of Oklahoma, George Lynn Cross Hall, 770 Van Vleet Oval, Norman, OK 73019, USA. Tel.: +1-405-325-0437; fax: +1-405-325-7619. E-mail address: [email protected] (L.R. Krumholz). 1 Present address: Water Environment and Remediation Center, KIST, 39-1, Hawolgok-dong, Sungpook-ku, Seoul 136-791, Korea.
Following the RT reaction, a second arbitrary primer is added to the reaction mixture and double stranded cDNAs are amplified by conventional PCR under low stringency conditions. As prokaryotic mRNA does not have the poly(A) tail, arbitrary primers are typically used for both the RT and PCR steps (McClelland et al., 1997; Rothschild et al., 1997), and this process is termed random arbitrarily primed (RAP) PCR. PCR products are visualized after separation on denaturing polyacrylamide gels, and profiles of cells grown under different conditions are compared. Unique RNAs are then cloned and sequenced. Although these methods have been widely used because of their sensitivity and relative low cost, problems have been reported, which include false positives (Faslage et al., 1997; Nagel et al., 1999) and occasional irreproducibility (Liang and Pardee, 1992;
0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00093-9
428
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
Mou et al., 1994). In addition, even if the amplified product is exactly cut out, the PCR product may be a mixture of products of similar size (Mathieu-Daude et al., 1996). Here we report that a fraction of false positive bands, originally identified as rRNA products, are actually chimeric sequences some of which are composed of rRNA and a differentially expressed gene product (mRNA). Our specific interests are in the response of sulfatereducing bacteria (SRB) to metals at the genetic level. SRB are important microorganisms in a variety of anaerobic ecosystems, including those contaminated with metals (Sani et al., 2001). Several metal bioremediation approaches, in situ (Waybrant et al., 1998) and bioreactor (Chang et al., 2000; White and Gadd, 1996) based, have been proposed, which involved SRB. We have attempted to identify genes expressed in response to metals using random arbitrarily primed polymerase chain reaction (RAP-PCR). Desulfovibrio vulgaris Hildenborough was used as the model strain throughout the study, and Cu(II) was used as a heavy metal. D. vulgaris was cultivated anaerobically under fermentative conditions in a mineral medium containing 0.2% yeast extract, 0.0001% resazurin (redox indicator), vitamins, minerals, and trace metals prepared as previously described (Krumholz and Bryant, 1986). The medium contained 20 mM pyruvate as electron acceptor and was treated with/without 50 AM CuCl2. When cultures were in early log phase, total RNA was isolated as described by Steger et al. (2001). RAP-PCR was then performed as described by Shepard and Gilmore (1999). The sequences of the arbitrary primers were AATCTAGAGCTCTCCTGG (primer 1) and AATCTAGAGCTCCCTCCA (primer 2). Products were labeled with 33P-dCTP and visualized by running on a denaturing polyacrylamide gel (6%) at 1,200 V for 10 h. The gel was dried and exposed to Kodak BioMax MR film as well as a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA USA) for 18 h at room temperature. Fig. 1 shows the cDNA profiles of RAPPCR. The two candidate bands (band #1 and band #2) were excised from the gel, eluted with buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS, precipitated with 100% ethanol, and resuspended in 10 Al sterile water. The cDNA was then reamplified and resolved on an identical gel loaded adjacent to the original RAP-PCR product to ensure that the correct band was reamplified
Fig. 1. cDNA band profiles on denaturing gels after RAP-PCR using primer 2. Lanes 1 – 7: 50 AM CuCl2 treatment and lanes 8 – 13, control (no treatment).
(data not shown). The candidate PCR product was ligated into pCR4-TOPO vector (Invitrogen, Carlsbad, CA, USA) and transformed into chemically competent One Shot TOP10 E. coli (Invitrogen). Plasmid was isolated from 3 ml of liquid cultures of candidate clones grown in LB + ampicillin (100 Ag/ml) using a plasmid isolation kit (Qiaprep Spin Miniprepkit, QIAGEN, Valenica, CA, USA) according to the manufacturer’s directions. After plasmid isolation, the plasmids were cut with EcoR1 (Invitrogen) and resolved on a
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
0.8% (w/v) agarose gel to compare inserts (data not shown). DNA sequencing was carried out at the Oklahoma Medical Research Foundation (Oklahoma City, OK) Core Sequencing Facility. For this study, we cloned 8 bands and picked 10 clones to be sequenced per transformation. Candidate insert sequences were compared to the whole genome of D. vulgaris Hildenborough in TIGR BLAST Search Engine for Unfinished Microbial Genomes (UFMG). Fig. 2 shows an example sequence of band #1 that contains sequences from two different open reading frames but shared an identical DNA sequence of 29 base pairs. These sequences were found within the genomic sequence of D. vulgaris but were separated by about 300 kb of genomic DNA. This type of result occurred in an average of 5 of every 10 clones sequenced and occurred
429
with both of the tested arbitrary primers. Of the 36 chimeric sequences observed, 3 contained chimeras of mRNA and rRNA, and the remainder were of 2 different rRNA sequences. The remaining nonchimeric sequences were all of 16S or 23S rRNA. In order to be certain that the observed sequences did not occur as a result of genetic recombination within the sulfate-reducing bacterium, we designed primer sets (boxed sequences in Fig. 2A) to amplify each of the two genes on a chimeric sequence and amplified chromosomal and plasmid DNA as templates. Chromosomal DNA was isolated using Easy-DNAk kit (Invitrogen) according to the manufacturer’s directions. To initiate PCR experiments, 1 Ag chromosomal DNA or cloned plasmid DNA was added to a 0.65 ml thin-wall PCR tube with 5 Al buffer (10X buffer,
Fig. 2. The chimeric sequence (A) and schematic of TIGR whole genome search results (B) shown in the same order. The sequence was obtained from one of the clones (band #1 in Fig. 1), and a whole genome search was performed using Unfinished Microbial Genome (UFMG) in TIGR (http://tigrblast.tigr.org/ufmg). Specific primer sets (indicated by boxed regions) were designed to reamplify each gene of the chimeric sequence for which results are presented in Fig. 3. The underlined sequence is identical to that present in one of the genes and similar to that present in the second gene. Random primer sequence is not shown.
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I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
Invitrogen), 1 Al dNTP (10 mM), 1.5 Al MgCl2 (50 mM), 1 Al primer mixture (50 mM of each forward and reverse primer), 0.5 Al Taq polymerase (5 U/Al, Invitrogen), and the total volume was adjusted to 50 Al with water. The reaction mixture was incubated at 95 jC for 10 min, and the following parameters were used for an additional 30 cycles of PCR: 94 jC (1 min), 56 jC (1 min), 72 jC (1 min), and a final extension at 72 jC for 10 min. Fig. 3A shows the 1.5% agarose gel electrophoresis (120 V for 30 min) results of band #1. The predicted PCR product size for the two individual open reading frames were observed with both chromosomal
Fig. 3. Electrophoresis results confirming the chimeric sequence in clones. The chimeric sequence obtained from band #1 (A) and band #2 (B). The predicted PCR product size for the two individual open reading frames were observed with both cloned (lanes 1 and 2) and chromosomal (lanes 4 and 5) DNA. The PCR product of the chimeric sequence was observed with the cloned DNA (lane 6) but not with the chromosomal DNA (lane 3).
(lanes 4 and 5) and cloned (lanes 1 and 2) DNA (Fig. 3A). However, when we attempted to amplify the entire chimeric sequence, a product was observed with the cloned DNA (lane 6) but not with chromosomal DNA (lane 3). Other chimeric sequences (band #2) were examined with similar results (Fig. 3). Because only one random primer is used for both first (RT reaction) and second strand synthesis, the cloned sequences are flanked by the same primer sequence. The primer sequence was not observed in the middle of the cloned sequence. However, when chimeric sequences are used to search the whole genome (UFMG) D. vulgaris database, we always observed a region where sequence identity or high homology exists between the two sequences within the chimera. In D. vulgaris, the whole genome has five copies of 23S and 16S rDNA located throughout the genome. As shown in Fig. 2B, the chimeric sequence has a sequence derived from 23S rRNA and another sequence that could be derived from mRNA. This result indicates that even if RAP-PCR products have a high similarity to rRNA sequence after BLAST searches, it is possible that they could contain important information. In fact, we have shown that some of the mRNA sequences within the chimeras are from induced mRNAs (data not shown). The occurrence of chimeric clones during more traditional PCR based cloning experiments is much more rare (Speksnijder et al., 2001) than we have observed using RAP-PCR. The major difference being that with RAP-PCR, RNA is the template and random primers are being used. A common feature of chimeric sequences is the sequence near the middle common to both rRNA and mRNA or to two rRNAs. This region ranged in size from 9 to 64 base pairs in our clones. Typically, the common sequence shows identity to one sequence from the database and similarity to the second sequence. Because of this feature and based on the relative abundance of chimeric clones, we suggest that chimeras likely occur as two cDNAs may form a duplex during the early stage of the process. Random primers bind to only one site on each of the strands and likely amplify the entire duplex. One of the major problems with DD- or RAP-PCR techniques is the high incidence of false positive bands, which turn out to have rRNA sequences (Faslage et al., 1997; Nagel et al., 1999, 2001). This is typically attributed to the relative abundance of rRNA in the total RNA pool. Several research groups
I.S. Chang et al. / Journal of Microbiological Methods 54 (2003) 427–431
have attempted to minimize these false results using random primers specifically designed to avoid rRNA amplification (Faslage et al., 1997; Nagel et al., 1999). However, this method is likely to be of limited use because of the requirement for genomic sequence information. Recently, Nagel et al. (2001) described a screening method using oligonucleotide probes designed to identify and ultimately eliminate clones derived from rRNAs. Colony blots were prepared and those colonies, which did not hybridize to rRNA probes, were used to identify genes in Pseudomonas expressed in response to Cadmium exposure. Although these approaches could be used to reduce the incidence of false positive results, our experiments demonstrated that rRNA containing sequences often contain useful information. As rRNA containing chimeric sequences was observed in mRNA containing clones, it is unwise to eliminate them. Because RAPPCR has been widely used to identify transcripts differentially expressed under altered conditions, this finding could be useful for interpretation of data from DD- and RAP-PCR.
Acknowledgements This research was supported by a grant from the Natural and Accelerated Bioremediation Research (NABIR) program of the Office of Biological and Environmental Research of the U.S. Department of Energy, Office of Science.
References Chang, I.S., Shin, P.K., Kim, B.H., 2000. Biological treatment of acid mine drainage under sulphate-reducing conditions with solid waste materials as substrate. Water Res. 34, 1269 – 1277. Faslage, R., Berceanu, M., Humboldt, Y., Wendt, M., Oberender, H., 1997. Primer design for a prokaryotic differential display RT-PCR. Nucleic Acids Res. 25, 1830 – 1835. Krumholz, L.R., Bryant, M.P., 1986. Eubacterium oxidoreducens
431
sp. nov. requiring H2 or formate to degrade gallate, pyrogallol, phloroglucinol and quercetin. Arch. Microbiol. 144, 8 – 14. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967 – 971. Mathieu-Daude, F., Cheng, R., Welsh, J., McClelland, M., 1996. Screening of differently amplified cDNA products from RNA arbitrarily primer PCR fingerprints using single strand conformation polymorphism (SSCP) gels. Nucleic Acids Res. 24, 1504 – 1507. McClelland, M., Honeycutt, R., Mathieu-Daude, F., Vogt, T., Welsh, J., 1997. Finger printing by arbitrarily primer PCR. Methods Mol. Biol. 85, 13 – 24. Mou, L., Miller, H., Li, J., Wang, E., Chalofour, L., 1994. Improvements to the differential display method for gene analysis. Biochem. Biophys. Res. Commun. 199, 564 – 569. Nagel, A., Fleming, J.T., Sayler, G.S., 1999. Reduction of false positives in prokaryotic mRNA differential display. BioTechniques 26, 641 – 648. Nagel, A.C., Fleming, J.T., Sayler, G.S., Beattie, K.L., 2001. Screening for ribosomal-based false positives following prokaryotes mRNA differential display. BioTechniques 30, 988 – 996. Rothschild, D.B., Brewer, C.S., Bowden, D.W., 1997. DD/APPCR: combination of differential display and arbitrarily primed PCR of oligo (dt) cDNA. Anal. Biochem. 245, 48 – 54. Sani, R.K., Peyton, B.M., Brown, L.T., 2001. Copper-induced inhibition of growth of Desulfovibrio desulfuricans G20: assessment of its toxicity and correlation with those of zinc and lead. Appl. Environ. Microbiol. 67, 4765 – 4772. Shepard, B., Gilmore, M., 1999. Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR. Appl. Environ. Microbiol. 65, 1470 – 1476. Speksnijder, A.G.C.L., Kowalchuk, G.A., De Jong, S., Kline, E., Stephen, J.R., Laanbroek, H.J., 2001. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl. Environ. Microbiol. 67, 469 – 472. Steger, J.L., Vincent, C., Ballard, J.D., Krumholz, L.R., 2001. Desulfovibrio sp. genes involved in the respiration of sulfate during metabolism of hydrogen and lactate. Appl. Environ. Microbiol. 68, 1932 – 1947. Waybrant, K.R., Blowes, D.W., Ptacek, C.J., 1998. Selection of reactive mixtures for use in permeable reactive walls for treatment of mine drainage. Environ. Sci. Technol. 32, 1972 – 1979. White, C., Gadd, G.M., 1996. Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration. Microbiology 142, 2197 – 2205.