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Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA
Gene 194 (1997) 273–276
Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA Robert S. Cormack, Imre E. Somssich * Max-Planck-Institut fu¨r Zu¨chtungsforschung, Abteilung Biochemie, Ko¨ln, Germany Received 12 December 1996; accepted 4 March 1997; Received by C.M. Kane
Abstract This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5∞ end of a gene, followed by denaturation and polyadenylation of its free 3∞ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3∞ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification. © 1997 Elsevier Science B.V. Keywords: Polymerase chain reaction; Terminal transferase; Gene; Restriction enzyme
1. Introduction Traditional approaches to the cloning of genomic sequences based upon cDNA sequence information involve the screening of lambda phage-generated libraries (Sambrook et al., 1989). Although effective, these methods consume considerable amounts of time, effort, and expense and normally require the use of radioisotopes. Since the introduction of the polymerase chain reaction (Saiki et al., 1988), several PCR-based cloning methods to isolate genomic DNA have been devised. These include inverse PCR (Ochman et al., 1988), ligation-mediated PCR (Mueller and Wold, 1991), Biotin-RAGE PCR (Bloomquist et al., 1992) and onearmed PCR (Macrae and Brenner, 1994). The degrees of difficulty and the effectiveness of these procedures are quite variable and do not always result in successful cloning of genomic sequences. We have developed a rapid method to clone upstream or downstream genomic * Corresponding author. Tel: +49 221 5062310; Fax: +49 221 5062313; e-mail: [email protected] Abbreviations: PCR, polymerase chain reaction; RAGE, rapid amplification of genomic ends; gDNA, genomic DNA; cDNA, DNA complementary to RNA; dNTP, deoxyribonucleoside triphosphate; bp, base pair. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 05 - 9
sequences following complementary DNA cloning procedures. Rapid amplification of cDNA ends, or RACE, has been used successfully to clone 5∞ and 3∞ cDNA ends (Ohara et al., 1989). RACE consists of the polynucleotidation of reverse-transcribed mRNA followed by amplification by PCR using gene-specific and anchored homooligomeric primers. Using a similar approach ( Fig. 1) we have been able to clone upstream genomic sequences from Arabidopsis and parsley based on downstream cDNA information.
2. Experimental and discussion
2.1. Polyadenylation and amplification of genomic DNA Approximately 1 mg of Arabidopsis thaliana genomic DNA was digested to completion with 80 units of EcoRI (Boehringer-Mannheim, Mannheim, Germany). The sample was extracted with an equal volume of phenol/chloroform/isoamyl alcohol and precipitated with the addition of 2.5 vol. 95% ethanol. The pellet was washed with 70% ethanol and resuspended in 10 ml of sterile distilled H O. The DNA was boiled for 5 min 2 then quick-cooled on ice. The entire sample was incu-
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R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
Fig. 1. Flow chart for rapid amplification of genomic ends.
bated with 0.5 mM dATP and 1.5 mM CoCl in 20 ml 2 of terminal transferase ( TdT ) buffer (BoehringerMannheim) containing 50 units of TdT at 37°C for 60–90 min. The reaction was stopped by heating the sample at 72°C for 5 min. The polymerase chain reaction was performed using 2 ml (100 ng) of the polyadenylated genomic DNA as template, 100 pmol of gene-specific Primer 1 (5∞-GCTTCCTTCTCATGAAGCTGGTTA), 100 pmol of universal-T17 primer (5∞-GTAAAACGACGGCCAGTCGACTTTTTTTTTTTTTTTTT ), 200 mM dNTPs and 5 units of Taq DNA polymerase (Boehringer-Mannheim) in 100 ml volume. The PCR was carried out at 94°C for 15 s, 60°C for 30 s, and 72°C for 1 min cycled 35 times using an OmniGene thermocycler (Hybaid Limited, Teddington, UK ). Following the reaction, 1 ml of the sample was then used as template for a second round of PCR under the same conditions, except that 100 pmol of gene-specific Primer 2 (5∞-TTGTTTCCAAGTAGCCTCAA) and 100 pmol of universal primer (5∞-GTAAAACGACGGCCAGT ) were used. A third PCR reaction was performed using 1 ml of product from the second reaction and the same primers and 2.5 units of Pfu DNA polymerase (Stratagene, Heidelberg, Germany) in order to generate large quantities of blunt-ended PCR product. The final PCR product was subcloned into the unique SmaI site of the vector pGFPuv (Clontech Laboratories, Palo Alto, CA, USA). The insert DNA was cycle-sequenced using an ABI377 (PE Applied Biosystems, Branchburg, NJ, USA) automated fluorescent sequencer. 2.2. Products obtained by PCR A 750-bp cDNA clone (At4) was originally isolated using the two-hybrid system while screening an
Arabidopsis oligo-dT-primed cDNA library. This cDNA sequence was used as the basis for testing a new PCRbased method to clone genomic ends (Fig. 1). An oligonucleotide (Primer 1) contiguous to the complementary strand of the At4 cDNA coding sequence situated approximately 560 bp downstream from an EcoRI restriction site was synthesized. It was used in conjunction with the universal-T17 primer and 100 ng of polyadenylated, EcoRI-digested Arabidopsis genomic DNA (see Section 2.1.) as template for PCR. Approximately 1% of the product was used as the template for a second PCR reaction using the universal primer and a second gene-specific primer (Primer 2) positioned about 475 bp upstream of, and in the same orientation as, Primer 1. The products from the first and second PCR reactions were separated in a 0.8% agarose gel ( Fig. 2). No detectable distinct bands were present from the first reaction ( lane 2), whereas a distinct band migrating at about 475 bp was seen from the second PCR reaction ( lane 3). The product was re-amplified using Pfu DNA polymerase, subcloned and sequenced. 2.3. Analysis of PCR-derived sequence The sequence of the original At4 cDNA and the sequence of the PCR amplified product (RAGE ) is shown in Fig. 3. The PCR-derived sequence is identical
Fig. 2. Gel electrophoresis of PCR products. Approximately 30 ml of each reaction was separated in an 0.8% (w/v) agarose gel containing 0.5 mg/ml ethidium bromide. Lane 1, kb ladder (BRL); lane 2, first PCR reaction; lane 3, second PCR reaction.
R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
275
Fig. 3. Sequence of At4 cDNA and RAGE-PCR product. A partial sequence of the cDNA is shown and the corresponding genomic sequence directly below. The sequences representing the universal-T17 primer and Primer 2 are underlined. The cDNA-imbedded EcoRI site is also underlined.
to that of the cDNA, but it also contains a 301-bp intron with canonical intron–exon boundaries, thus verifying that the PCR product is of genomic origin. Primer 2 spans an exon–intron border, but was still, however, able to anneal to its template because of a fortuitous repetition of a sequence in the gene that created the sequence to which the primer was designed (Fig. 3). The sequence contained 18 thymidine residues at its 5∞ end, indicating that the polyadenylation reaction produced a ‘tail’ of sufficient length. The sequence of the gene was verified. Arabidopsis genomic DNA was amplified using Primer 1 and a third oligonucleotide whose
sequence corresponds to that of the At4 cDNA just upstream of the EcoRI restriction site, subcloned and subjected to DNA sequencing (data not shown). It possessed 100% sequence identity to that of the RAGEPCR product. 2.4. Amplification of an Arabidopsis promoter The new method was used to amplify the 5∞ region of an Arabidopsis gene whose cDNA (At23) was originally isolated by conventional means. Initially, inverse PCR (Ochman et al., 1988) was performed to amplify a
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R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
1600-bp HindIII gDNA fragment using bi-directional primers specific for the At23 cDNA. The resulting gene sequence matched completely to that of the cDNA and housed an additional 500 bp of upstream sequence. Polyadenylated HindIII-digested Arabidopsis gDNA was used as the template for the RAGE-PCR amplification of the 5∞ promoter region of the At23 gene. Two genespecific primers, positioned about 650 bp and 560 bp downstream of the HindIII site, were used in the reactions. A product of about 700 bp was synthesized after the second PCR step, which was easily visualized in a ethidium bromide-stained agarose gel (data not shown). The DNA fragment was isolated, cloned and sequenced. The RAGE-PCR product exhibited 100% sequence identity to that of the inverse PCR product and possessed a poly(A) tail of about 50 bp (data not shown).
biotinylated primers or purification steps. Implementing the identical strategy, it should be possible to clone much more distant 5∞ or 3∞ genomic ends for a given cDNA by choosing a variety of restriction enzymes to digest the gDNA. In fact, a small collection of polyadenylated gDNA digested with various enzymes can be easily generated. However, in order to obtain an estimate of the expected final PCR product, a Southern blot (Brown, 1995) of single-enzyme digested gDNA is recommended. In addition, the gene-specific primers should be tested for their ability to amplify gDNA and not to lie on an intron–exon border. RAGE appears to be applicable to any gene, including those belonging to families, provided that gene-specific primers are utilized. Although this protocol functions well for both Arabidopsis and parsley, RAGE can be adapted for the amplification of genes from any genome.
2.5. Amplification of a parsley promoter The same RAGE protocol was applied to amplify 5∞ DNA sequences containing the promoter of a parsley (Petroselinum crispum) pathogenesis-related gene belonging to the PR-10 family of proteins (Rushton et al., 1996). Parsley genomic DNA digested with HindIII and EcoRI was used as template for the polyadenylation reaction. Two gene-specific primers complementary to the 5∞ region of a cDNA of a PR-10-type gene previously isolated (data not shown) were used to amplify the corresponding genomic DNA by the RAGE procedure. A PCR product of about 1100 bp was obtained and subjected to DNA sequence analysis. The PCR product possessed a long poly-A tail and about 975 bp of upstream sequence relative to the second nested primer (data not shown). There was complete sequence agreement between the 5∞ end of the cDNA clone (50 bp) and the corresponding gDNA, indicating that the correct genomic clone was generated. In addition, the genomic sequence exhibited 70–75% sequence identity to two other known members of parsley PR-10 gene family (Rushton et al., 1996).
3. Conclusions Rapid amplification of genomic ends (RAGE) can provide a quick and non-radioactive alternative to the cloning of genomic sequences. Its effective application was demonstrated by the cloning of two Arabidopsis sequences and a parsley promoter. Unlike previously described methods (e.g., Biotin-RAGE), the protocol does not require the use of radioisotopic detection,
Acknowledgement We would like to thank Andrea Hu¨sing for her contribution in the amplification of the parsley promoter. R.S.C. holds a Fellowship from the Alexandervon-Humboldt-Stiftung.
References Bloomquist, B.T., Johnson, R.C., Mains, R.E., 1992. Rapid isolation of flanking genomic DNA using Biotin-RAGE, a variation of singlesided polymerase chain reaction. DNA Cell Biol. 11, 791–797. Brown, T., 1995. Southern blotting. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Siedman, J.G., Smith, J.A., Struhl, K. (Eds.), Short Protocols in Molecular Biology, 3rd ed. Wiley, New York, NY, pp. 2.28–2.32. Macrae, A.D., Brenner, S., 1994. One armed PCR (OA-PCR): amplification of genomic DNA from a single primer domain. Genomics 24, 176–178. Mueller, P.R., Wold, B., 1991. Ligation-mediated PCR: applications to genomic footprinting. Methods 2, 20–31. Ochman, H., Gerber, A.S., Hartl, D.L., 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623. Ohara, O., Dorit, R.L., Gilbert, W., 1989. One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. USA 86, 5673–5677. Rushton, J.P., Torres, J.T., Parniske, M., Wernet, P., Hahlbrock, K., Somssich, I.E., 1996. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15, 5690–5700. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA Robert S. Cormack, Imre E. Somssich * Max-Planck-Institut fu¨r Zu¨chtungsforschung, Abteilung Biochemie, Ko¨ln, Germany Received 12 December 1996; accepted 4 March 1997; Received by C.M. Kane
Abstract This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5∞ end of a gene, followed by denaturation and polyadenylation of its free 3∞ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3∞ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification. © 1997 Elsevier Science B.V. Keywords: Polymerase chain reaction; Terminal transferase; Gene; Restriction enzyme
1. Introduction Traditional approaches to the cloning of genomic sequences based upon cDNA sequence information involve the screening of lambda phage-generated libraries (Sambrook et al., 1989). Although effective, these methods consume considerable amounts of time, effort, and expense and normally require the use of radioisotopes. Since the introduction of the polymerase chain reaction (Saiki et al., 1988), several PCR-based cloning methods to isolate genomic DNA have been devised. These include inverse PCR (Ochman et al., 1988), ligation-mediated PCR (Mueller and Wold, 1991), Biotin-RAGE PCR (Bloomquist et al., 1992) and onearmed PCR (Macrae and Brenner, 1994). The degrees of difficulty and the effectiveness of these procedures are quite variable and do not always result in successful cloning of genomic sequences. We have developed a rapid method to clone upstream or downstream genomic * Corresponding author. Tel: +49 221 5062310; Fax: +49 221 5062313; e-mail: [email protected] Abbreviations: PCR, polymerase chain reaction; RAGE, rapid amplification of genomic ends; gDNA, genomic DNA; cDNA, DNA complementary to RNA; dNTP, deoxyribonucleoside triphosphate; bp, base pair. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 2 05 - 9
sequences following complementary DNA cloning procedures. Rapid amplification of cDNA ends, or RACE, has been used successfully to clone 5∞ and 3∞ cDNA ends (Ohara et al., 1989). RACE consists of the polynucleotidation of reverse-transcribed mRNA followed by amplification by PCR using gene-specific and anchored homooligomeric primers. Using a similar approach ( Fig. 1) we have been able to clone upstream genomic sequences from Arabidopsis and parsley based on downstream cDNA information.
2. Experimental and discussion
2.1. Polyadenylation and amplification of genomic DNA Approximately 1 mg of Arabidopsis thaliana genomic DNA was digested to completion with 80 units of EcoRI (Boehringer-Mannheim, Mannheim, Germany). The sample was extracted with an equal volume of phenol/chloroform/isoamyl alcohol and precipitated with the addition of 2.5 vol. 95% ethanol. The pellet was washed with 70% ethanol and resuspended in 10 ml of sterile distilled H O. The DNA was boiled for 5 min 2 then quick-cooled on ice. The entire sample was incu-
274
R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
Fig. 1. Flow chart for rapid amplification of genomic ends.
bated with 0.5 mM dATP and 1.5 mM CoCl in 20 ml 2 of terminal transferase ( TdT ) buffer (BoehringerMannheim) containing 50 units of TdT at 37°C for 60–90 min. The reaction was stopped by heating the sample at 72°C for 5 min. The polymerase chain reaction was performed using 2 ml (100 ng) of the polyadenylated genomic DNA as template, 100 pmol of gene-specific Primer 1 (5∞-GCTTCCTTCTCATGAAGCTGGTTA), 100 pmol of universal-T17 primer (5∞-GTAAAACGACGGCCAGTCGACTTTTTTTTTTTTTTTTT ), 200 mM dNTPs and 5 units of Taq DNA polymerase (Boehringer-Mannheim) in 100 ml volume. The PCR was carried out at 94°C for 15 s, 60°C for 30 s, and 72°C for 1 min cycled 35 times using an OmniGene thermocycler (Hybaid Limited, Teddington, UK ). Following the reaction, 1 ml of the sample was then used as template for a second round of PCR under the same conditions, except that 100 pmol of gene-specific Primer 2 (5∞-TTGTTTCCAAGTAGCCTCAA) and 100 pmol of universal primer (5∞-GTAAAACGACGGCCAGT ) were used. A third PCR reaction was performed using 1 ml of product from the second reaction and the same primers and 2.5 units of Pfu DNA polymerase (Stratagene, Heidelberg, Germany) in order to generate large quantities of blunt-ended PCR product. The final PCR product was subcloned into the unique SmaI site of the vector pGFPuv (Clontech Laboratories, Palo Alto, CA, USA). The insert DNA was cycle-sequenced using an ABI377 (PE Applied Biosystems, Branchburg, NJ, USA) automated fluorescent sequencer. 2.2. Products obtained by PCR A 750-bp cDNA clone (At4) was originally isolated using the two-hybrid system while screening an
Arabidopsis oligo-dT-primed cDNA library. This cDNA sequence was used as the basis for testing a new PCRbased method to clone genomic ends (Fig. 1). An oligonucleotide (Primer 1) contiguous to the complementary strand of the At4 cDNA coding sequence situated approximately 560 bp downstream from an EcoRI restriction site was synthesized. It was used in conjunction with the universal-T17 primer and 100 ng of polyadenylated, EcoRI-digested Arabidopsis genomic DNA (see Section 2.1.) as template for PCR. Approximately 1% of the product was used as the template for a second PCR reaction using the universal primer and a second gene-specific primer (Primer 2) positioned about 475 bp upstream of, and in the same orientation as, Primer 1. The products from the first and second PCR reactions were separated in a 0.8% agarose gel ( Fig. 2). No detectable distinct bands were present from the first reaction ( lane 2), whereas a distinct band migrating at about 475 bp was seen from the second PCR reaction ( lane 3). The product was re-amplified using Pfu DNA polymerase, subcloned and sequenced. 2.3. Analysis of PCR-derived sequence The sequence of the original At4 cDNA and the sequence of the PCR amplified product (RAGE ) is shown in Fig. 3. The PCR-derived sequence is identical
Fig. 2. Gel electrophoresis of PCR products. Approximately 30 ml of each reaction was separated in an 0.8% (w/v) agarose gel containing 0.5 mg/ml ethidium bromide. Lane 1, kb ladder (BRL); lane 2, first PCR reaction; lane 3, second PCR reaction.
R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
275
Fig. 3. Sequence of At4 cDNA and RAGE-PCR product. A partial sequence of the cDNA is shown and the corresponding genomic sequence directly below. The sequences representing the universal-T17 primer and Primer 2 are underlined. The cDNA-imbedded EcoRI site is also underlined.
to that of the cDNA, but it also contains a 301-bp intron with canonical intron–exon boundaries, thus verifying that the PCR product is of genomic origin. Primer 2 spans an exon–intron border, but was still, however, able to anneal to its template because of a fortuitous repetition of a sequence in the gene that created the sequence to which the primer was designed (Fig. 3). The sequence contained 18 thymidine residues at its 5∞ end, indicating that the polyadenylation reaction produced a ‘tail’ of sufficient length. The sequence of the gene was verified. Arabidopsis genomic DNA was amplified using Primer 1 and a third oligonucleotide whose
sequence corresponds to that of the At4 cDNA just upstream of the EcoRI restriction site, subcloned and subjected to DNA sequencing (data not shown). It possessed 100% sequence identity to that of the RAGEPCR product. 2.4. Amplification of an Arabidopsis promoter The new method was used to amplify the 5∞ region of an Arabidopsis gene whose cDNA (At23) was originally isolated by conventional means. Initially, inverse PCR (Ochman et al., 1988) was performed to amplify a
276
R.S. Cormack, I.E. Somssich / Gene 194 (1997) 273–276
1600-bp HindIII gDNA fragment using bi-directional primers specific for the At23 cDNA. The resulting gene sequence matched completely to that of the cDNA and housed an additional 500 bp of upstream sequence. Polyadenylated HindIII-digested Arabidopsis gDNA was used as the template for the RAGE-PCR amplification of the 5∞ promoter region of the At23 gene. Two genespecific primers, positioned about 650 bp and 560 bp downstream of the HindIII site, were used in the reactions. A product of about 700 bp was synthesized after the second PCR step, which was easily visualized in a ethidium bromide-stained agarose gel (data not shown). The DNA fragment was isolated, cloned and sequenced. The RAGE-PCR product exhibited 100% sequence identity to that of the inverse PCR product and possessed a poly(A) tail of about 50 bp (data not shown).
biotinylated primers or purification steps. Implementing the identical strategy, it should be possible to clone much more distant 5∞ or 3∞ genomic ends for a given cDNA by choosing a variety of restriction enzymes to digest the gDNA. In fact, a small collection of polyadenylated gDNA digested with various enzymes can be easily generated. However, in order to obtain an estimate of the expected final PCR product, a Southern blot (Brown, 1995) of single-enzyme digested gDNA is recommended. In addition, the gene-specific primers should be tested for their ability to amplify gDNA and not to lie on an intron–exon border. RAGE appears to be applicable to any gene, including those belonging to families, provided that gene-specific primers are utilized. Although this protocol functions well for both Arabidopsis and parsley, RAGE can be adapted for the amplification of genes from any genome.
2.5. Amplification of a parsley promoter The same RAGE protocol was applied to amplify 5∞ DNA sequences containing the promoter of a parsley (Petroselinum crispum) pathogenesis-related gene belonging to the PR-10 family of proteins (Rushton et al., 1996). Parsley genomic DNA digested with HindIII and EcoRI was used as template for the polyadenylation reaction. Two gene-specific primers complementary to the 5∞ region of a cDNA of a PR-10-type gene previously isolated (data not shown) were used to amplify the corresponding genomic DNA by the RAGE procedure. A PCR product of about 1100 bp was obtained and subjected to DNA sequence analysis. The PCR product possessed a long poly-A tail and about 975 bp of upstream sequence relative to the second nested primer (data not shown). There was complete sequence agreement between the 5∞ end of the cDNA clone (50 bp) and the corresponding gDNA, indicating that the correct genomic clone was generated. In addition, the genomic sequence exhibited 70–75% sequence identity to two other known members of parsley PR-10 gene family (Rushton et al., 1996).
3. Conclusions Rapid amplification of genomic ends (RAGE) can provide a quick and non-radioactive alternative to the cloning of genomic sequences. Its effective application was demonstrated by the cloning of two Arabidopsis sequences and a parsley promoter. Unlike previously described methods (e.g., Biotin-RAGE), the protocol does not require the use of radioisotopic detection,
Acknowledgement We would like to thank Andrea Hu¨sing for her contribution in the amplification of the parsley promoter. R.S.C. holds a Fellowship from the Alexandervon-Humboldt-Stiftung.
References Bloomquist, B.T., Johnson, R.C., Mains, R.E., 1992. Rapid isolation of flanking genomic DNA using Biotin-RAGE, a variation of singlesided polymerase chain reaction. DNA Cell Biol. 11, 791–797. Brown, T., 1995. Southern blotting. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Siedman, J.G., Smith, J.A., Struhl, K. (Eds.), Short Protocols in Molecular Biology, 3rd ed. Wiley, New York, NY, pp. 2.28–2.32. Macrae, A.D., Brenner, S., 1994. One armed PCR (OA-PCR): amplification of genomic DNA from a single primer domain. Genomics 24, 176–178. Mueller, P.R., Wold, B., 1991. Ligation-mediated PCR: applications to genomic footprinting. Methods 2, 20–31. Ochman, H., Gerber, A.S., Hartl, D.L., 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623. Ohara, O., Dorit, R.L., Gilbert, W., 1989. One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. USA 86, 5673–5677. Rushton, J.P., Torres, J.T., Parniske, M., Wernet, P., Hahlbrock, K., Somssich, I.E., 1996. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15, 5690–5700. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.