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Rapid Generation of Vector-Free Digoxygenin-dUTP Labeled Probes for Nonradioactive Hybridization Using the Polymerase Chain Reaction (PCR) Method
System. App!. Microbio!. 13,255-256 (1990) © Gustav Fischer Verlag, StuttgartlNew York
Short Communication
Rapid Generation of Vector-Free Digoxygenin-dUTP Labeled Probes for Nonradioactive Hybridization Using the Polymerase Chain Reaction (PCR) Method WERNER LIESACK, MARTIN A. O. H. MENKE, and ERKO STACKEBRANDT* Institut fur Allgemeine Mikrobiologie, ehristian-Albrechts-Universitat, 2300 Kiel, Federal Republic of Germany
Received January 17, 1990
During the last few years the search for replacement of radioactive labeling of nucleic acids for gene analysis has led to several successful alternatives, such as the biotin! streptavidin system, the modification of DNA by insertion of an antigenic sulfone group, or the incorporation of a nucleotide analogue into DNA by random priming. Here we present a one-step procedure based on the PCR method for synthesis and labeling of vector-free DNA probes with digoxygenin-dUTP (dig-dUTP) for non-radioactive hybridization. For unlabeled probes, PCR was done as described by Saiki et al. (1988), using 20 nmol of each dNTP and 100 pmol of each primer. For labeling the amount of dITP was reduced to 18 nmol and 2 nmol dig-dUTP (Boehringer, Mannheim, FRG) added. 0.5 Ilg genomic DNA from E. coli was used for amplifying a stretch of 16S rONA. The 5' and 3' primers (produced on an automated DNA synthesizer, Applied Biosystem, model 381A) were TCCTACGGGAGGCAGCAG and GGCGGTGTGTACAAGGCCC, respectively. These primers, which are universally applyable to eubacterial 16S rONA genes, allow the Taq polymerase to synthesize a stretch of 1063 bp between positions 340 and 1403 (IUB nomenclature of E. coli). Thirty cycles (annealing for 2 min at 50 elongation for 2.5 min at 72 denaturation for 1 min at 93 0c) were carried out in a Biozym DNA Incubator II. After 30 cycles 2-3 Ilg amplified rONA were obtained. Similar yields were found for labeled and non-labeled samples. Amplified products were purified by agarose gel electrophoresis (1.0%) and isolated by NA-45 DEAE membrane according to the supplier's protocol (Schleicher and
ec,
ec,
* Present address: Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia 18 System. Appl. Microbial. Vol. 13/3
Schull, 3354 Dassel, FRG). As already described for biotinylated PCR products (La et al., 1988) the nonlabeled reference sample migrated slightly faster than the dig-dUTP labeled probe (Fig. 1). Hybridization with 100 ng probe/l00 cm3 filter area were performed according to the protocol of the dig-dUTP DNA labeling and detection kit from Boehringer, Mannheim. Hybridization targets were BamHI digested genomic DNA of E. coli and the cloned 16S rONA of the rrnB operon of E. coli located on a 7.5 kb BamHI insert of pKK3535 (Brosius et al., 1978). DNA was blotted (Southern, 1975) on Hybond N membrane (Amersham, UK). Under the conditions applied, 10 pg of 1 kb target DNA can be detected (Fig. 2). This is sufficient for screening a single copy gene contained in 100 ng procaryotic genomic DNA. The amount of labeled probe used in the hybridization assay can most likely be further reduced by using a probe with higher specific activity. This can possibly be achieved by increasing the ratio of dig-dUTP/dNTP to a
164kb102 kbFig. 1. Ethidium-bromide stained DNA after electrophoresis on 3% NuSieve Gel (FMC); A, 1 kb-ladder (BRL) as size marker. B, amplified non-labeled reference sample. e, dig-dUTP labeled peR product.
256
W.Liesack, M.A.O.H.Menke, and E.Stackebrandt
B
A
o
c
E
Fig. 2. Determination of the sensitivity of duplexes formed by various amounts of target DNA and 100 ng dig-dUTP labeled 16S rDNA probe. A, 500 ng BamHl digested genomic DNA of E. coli; the 7 rDNA operons resolve in 3 bands (developed for 2 h). B-E, various amounts of BamHl digested plasmid pKK 3535. B, 10 ng (developed for 2 h), the weak signal below the rDNA fragment is the undigested ccc-form of pKK 3535. C, 1 ng (2 h). D, 100 pg (4 h). E, 10 pg (10 h).
Acknowledgement. This work was supported by a grant from the DFVLR (01 Kl 8836)
.
-
level indicated for labeling DNA during random priming (Boehringer Detection and Labeling Protocol, 1989). The combination of the PCR method and labeling with digdUTP for non-radioactive hybridizations is a convenient method for the rapid production of vector-free homologous DNA probes. Most promissing can this system be used in studies on the detection of rRNAs and rRNA genes where information on conservative sequences is available, necessary for designing probes at all levels of taxonomic ranks. Complete 16S rRNA genes have been successfully amplified (using primers GAGUUUGAU-C(C/A)UGGCUCAG and AGAAAGGAGGTGATCCA(AlG/CI T)CC), labeled with dig-dUTP and subsequently used as a probe in Southern hybridization for the determination of the number of 16S rRNA genes in Thermotoga maritima and Verrucomicrobium spinosum (Fig. 3).
-
A B CDEFGH
.
References
Brosius, ]., Ullrich, A., Raker, M. A., Gray, A., Dull, T. j., Gutell, R. R., Noller, H. F.: Construction and fine mapping of
recombinant plasmids containing the rnnB ribosomal RNA operon of E. coli. Plasmid 6, 112-118 (1981) Lo, Y.-M. D., Mehal, W. Z., Fleming, K. A.: Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids. Res. 16, 8719 (1988)
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. ]. Higuchi, R., Horn, G. T., Mullis, K. B., Ehrlich, H. A.: Primer-directed
a
b
Fig. 3. Southern hybridization of restricted DNA from Thermotoga maritima (a) and Verrucomicrobium spinosum (b) with amplified and dig-dUTP labeled 16S rDNA from E. coli as a probe. A, Xhol; B, Sail; C, Sad; D, PstI; E, HindIII; F, Eco Rl; G, Clal; H, Bcll. Fragments were separated on 0.5% agarose by FIGE and blotted to Hybond N. The background pattern in Fig. 3b results from N'N' -dimethylformanid treatment during decolorization for rehybridization (Boehringer dig-dUTP protocol). Hybridization temperature was 62°C, all other steps were performed as indicated by the manufacturer. Development was for 10h.
enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491 (1988) Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Molec. BioI. 98, 503-517 (1975)
Dr. Werner Liesack, lnstitut fur Allgemeine Mikrobiologie der Universitiit, Olshausenstr. 40, D-2300 Kiel
Short Communication
Rapid Generation of Vector-Free Digoxygenin-dUTP Labeled Probes for Nonradioactive Hybridization Using the Polymerase Chain Reaction (PCR) Method WERNER LIESACK, MARTIN A. O. H. MENKE, and ERKO STACKEBRANDT* Institut fur Allgemeine Mikrobiologie, ehristian-Albrechts-Universitat, 2300 Kiel, Federal Republic of Germany
Received January 17, 1990
During the last few years the search for replacement of radioactive labeling of nucleic acids for gene analysis has led to several successful alternatives, such as the biotin! streptavidin system, the modification of DNA by insertion of an antigenic sulfone group, or the incorporation of a nucleotide analogue into DNA by random priming. Here we present a one-step procedure based on the PCR method for synthesis and labeling of vector-free DNA probes with digoxygenin-dUTP (dig-dUTP) for non-radioactive hybridization. For unlabeled probes, PCR was done as described by Saiki et al. (1988), using 20 nmol of each dNTP and 100 pmol of each primer. For labeling the amount of dITP was reduced to 18 nmol and 2 nmol dig-dUTP (Boehringer, Mannheim, FRG) added. 0.5 Ilg genomic DNA from E. coli was used for amplifying a stretch of 16S rONA. The 5' and 3' primers (produced on an automated DNA synthesizer, Applied Biosystem, model 381A) were TCCTACGGGAGGCAGCAG and GGCGGTGTGTACAAGGCCC, respectively. These primers, which are universally applyable to eubacterial 16S rONA genes, allow the Taq polymerase to synthesize a stretch of 1063 bp between positions 340 and 1403 (IUB nomenclature of E. coli). Thirty cycles (annealing for 2 min at 50 elongation for 2.5 min at 72 denaturation for 1 min at 93 0c) were carried out in a Biozym DNA Incubator II. After 30 cycles 2-3 Ilg amplified rONA were obtained. Similar yields were found for labeled and non-labeled samples. Amplified products were purified by agarose gel electrophoresis (1.0%) and isolated by NA-45 DEAE membrane according to the supplier's protocol (Schleicher and
ec,
ec,
* Present address: Department of Microbiology, University of Queensland, St. Lucia, Queensland 4067, Australia 18 System. Appl. Microbial. Vol. 13/3
Schull, 3354 Dassel, FRG). As already described for biotinylated PCR products (La et al., 1988) the nonlabeled reference sample migrated slightly faster than the dig-dUTP labeled probe (Fig. 1). Hybridization with 100 ng probe/l00 cm3 filter area were performed according to the protocol of the dig-dUTP DNA labeling and detection kit from Boehringer, Mannheim. Hybridization targets were BamHI digested genomic DNA of E. coli and the cloned 16S rONA of the rrnB operon of E. coli located on a 7.5 kb BamHI insert of pKK3535 (Brosius et al., 1978). DNA was blotted (Southern, 1975) on Hybond N membrane (Amersham, UK). Under the conditions applied, 10 pg of 1 kb target DNA can be detected (Fig. 2). This is sufficient for screening a single copy gene contained in 100 ng procaryotic genomic DNA. The amount of labeled probe used in the hybridization assay can most likely be further reduced by using a probe with higher specific activity. This can possibly be achieved by increasing the ratio of dig-dUTP/dNTP to a
164kb102 kbFig. 1. Ethidium-bromide stained DNA after electrophoresis on 3% NuSieve Gel (FMC); A, 1 kb-ladder (BRL) as size marker. B, amplified non-labeled reference sample. e, dig-dUTP labeled peR product.
256
W.Liesack, M.A.O.H.Menke, and E.Stackebrandt
B
A
o
c
E
Fig. 2. Determination of the sensitivity of duplexes formed by various amounts of target DNA and 100 ng dig-dUTP labeled 16S rDNA probe. A, 500 ng BamHl digested genomic DNA of E. coli; the 7 rDNA operons resolve in 3 bands (developed for 2 h). B-E, various amounts of BamHl digested plasmid pKK 3535. B, 10 ng (developed for 2 h), the weak signal below the rDNA fragment is the undigested ccc-form of pKK 3535. C, 1 ng (2 h). D, 100 pg (4 h). E, 10 pg (10 h).
Acknowledgement. This work was supported by a grant from the DFVLR (01 Kl 8836)
.
-
level indicated for labeling DNA during random priming (Boehringer Detection and Labeling Protocol, 1989). The combination of the PCR method and labeling with digdUTP for non-radioactive hybridizations is a convenient method for the rapid production of vector-free homologous DNA probes. Most promissing can this system be used in studies on the detection of rRNAs and rRNA genes where information on conservative sequences is available, necessary for designing probes at all levels of taxonomic ranks. Complete 16S rRNA genes have been successfully amplified (using primers GAGUUUGAU-C(C/A)UGGCUCAG and AGAAAGGAGGTGATCCA(AlG/CI T)CC), labeled with dig-dUTP and subsequently used as a probe in Southern hybridization for the determination of the number of 16S rRNA genes in Thermotoga maritima and Verrucomicrobium spinosum (Fig. 3).
-
A B CDEFGH
.
References
Brosius, ]., Ullrich, A., Raker, M. A., Gray, A., Dull, T. j., Gutell, R. R., Noller, H. F.: Construction and fine mapping of
recombinant plasmids containing the rnnB ribosomal RNA operon of E. coli. Plasmid 6, 112-118 (1981) Lo, Y.-M. D., Mehal, W. Z., Fleming, K. A.: Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids. Res. 16, 8719 (1988)
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. ]. Higuchi, R., Horn, G. T., Mullis, K. B., Ehrlich, H. A.: Primer-directed
a
b
Fig. 3. Southern hybridization of restricted DNA from Thermotoga maritima (a) and Verrucomicrobium spinosum (b) with amplified and dig-dUTP labeled 16S rDNA from E. coli as a probe. A, Xhol; B, Sail; C, Sad; D, PstI; E, HindIII; F, Eco Rl; G, Clal; H, Bcll. Fragments were separated on 0.5% agarose by FIGE and blotted to Hybond N. The background pattern in Fig. 3b results from N'N' -dimethylformanid treatment during decolorization for rehybridization (Boehringer dig-dUTP protocol). Hybridization temperature was 62°C, all other steps were performed as indicated by the manufacturer. Development was for 10h.
enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491 (1988) Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Molec. BioI. 98, 503-517 (1975)
Dr. Werner Liesack, lnstitut fur Allgemeine Mikrobiologie der Universitiit, Olshausenstr. 40, D-2300 Kiel