- Email: [email protected]
Direct characterization, enrichment and sequencing of differential displayed cDNA sequences
Technical Tips Online, Vol. 3, 1998
Direct characterization, enrichment and sequencing of differential displayed cDNA sequences Jin Zenga,b, Mingfang Liua and Jean-Charles Frucharta a b
Institute Pasteur de Lille, 1 Rue Calmette, 59019 Lille, France NIMH/NIH, Building 10, Room 3N218, Bethesda, MD 20892, USA
Keywords: Polymerase chain reaction, Cloning & sequencing ▼Differential display of mRNA by PCR (DD-PCR) is a popular technique for cloning selectively expressed genes (Ref. 1). This method has some unique advantages over classical differential cloning methods (Ref. 2), such as the possibility for simultaneous comparison of mRNA differences among several samples. Its principle is elegantly simple; nevertheless, there are several practical difficulties with this technique. Most notably, owing to the fact that a significant number of differential fragments displayed as a single band on sequencing gels are heterogeneous (Ref. 3, 4, 5), true positive cDNAs must be differentiated from contaminating sequences by selection. Several methods for the postdisplay screening have been reported (Ref. 6, 7); however, their main utility is limited to identify distinct inserts from cloning. A complete post-display analysis often involves screening hundreds of clones for a handful of distinct and desirable cDNAs. It is a major bottleneck of differential display and has been recognized as among the most cumbersome steps of the method (Ref. 8, 9). Furthermore, it can be prohibitively expensive (Ref. 10). Here we present a rapid and cost-effective approach without recourse to cloning for direct characterization of differential cDNA molecules, up to the point of sequence identification. The experimental conditions have been optimized so that the protocol could be reliably performed with only three steps of minimal manipulation. It has the following novel features and advantages. First, our method of direct separation is very economical and effective. Like Geisinger et al. (Ref. 7, 11), we also use
the DNA ligand bisbenzimide to separate co-migrating sequences. However, our method does not involve cloning. As expected from the fact that differential cDNA sequences are mainly derived from AT-rich 3 -UTR regions of mRNA molecules to which bisbenzimide preferentially binds, major components of a vast majority of reamplified differential molecules could be directly separated by the DNA dye using the electrophoresis conditions stated below (data not shown). Second, cDNA fragments resolved by bisbenzimide can be directly enriched by PCR without prior purification. We observed that, by contrast with other DNA ligands such as isopsoralen (Ref. 12), bisbenzimide could not photochemically block amplification by Taq polymerase under the conditions (data not shown and see the protocol). Thus, no labor-intensive manipulation for cleaning up the PCR products is needed when using the procedure. Third, our method of separation and enrichment is highly efficient. The differential cDNA fragments recovered as such are usually so pure that they could even be reliably used in automatic sequencing under the optimized cycle reaction condition presented below.
Protocol 1.
2. Corresponding author: [email protected]
c 1366-2120 1998 Elsevier Ltd. All rights reserved. PII: S1366-2120(08)70115-6
Carry out differential display and reamplify differential bands (their sizes should optimally be more than 200 bp) after excising them from gel using the protocols of Linskens et al. (Ref. 13) or the secondgeneration RNAimage kit (GenHunter). Prepare a 2% agarose gel with bisbenzimide-PEG 6000 [‘Resolver GoldTM ’, (R&D System)] by adding 1 unit of
www.sciencedirect.com
109
Technical Tips Online, Vol. 3, 1998
3. 4. 5. 6. 7. 8.
9.
10.
11.
the agent per ml into agarose gel precooled to 70◦ C or less. Use 1×TAE, pH7.2 as gel and electrophoresis buffers (note that pH of the buffer is critical!). Load 20 µl of the reamplified differential cDNA samples with bromophenol blue. Run the electrophoresis at a voltage of 5 Vcm−1 until the dye reaches about two third of the gel. Stain the gel with 0.5 µg/ml of ethidium bromide. Visualize the bands using a UV lamp and remove them into an Eppendorf tube containing 100 µl of H2 O. Heat the gel band at 100◦ C for 5 min. Use 2 µl of the gel solution for PCR amplification: 4 µl 10×PCR buffer, 4 µl 25 mM MgCl2 , 3.2 µl 0.25 mM dNTP, 1.6 µl 10 mM 5 and 3 primers, 0.4 µl Taq polymerase and water to 40 µl. Carry out 30 cycles (94◦ C for 50 s; 60◦ C for 60 s; and 72◦ C for 60 s) with a final extension at 72◦ C for 10 min. Upon amplification, load 10 µl of the product onto a normal agarose gel for verification, if necessary. Pretreat the PCR amplified samples for sequencing: add 0.5 µl (10 U/µl) Exonuclease I (Amersham) and 0.5 µl (2 U/µl) Shrimp alkaline phosphatase (Amersham) into 2.5 µl of the re-amplified product in a PCR tube; incubate the reaction mixture at 37◦ C for 30 min, and then at 80◦ C for 15 min. Carry out the cycle sequencing reaction: add 0.5 µl 10 µM 5 random primer, 8µl ABI Mix (Perkin-Elmer), 8 µl H2 O directly into the pretreated reaction mixture. Carry out 25 cycles with an initial incubation at 96◦ C for 10 s (96◦ C, 10 s; 56◦ C, 5 s; 60◦ C, 240 s). Precipitate the sequencing reaction by ethanol as recommended by the manufacturer (Perkin-Elmer) and analyze the sequence using an ABI prism 377 sequencer (Perkin-Elmer). Note that manual sequencing could also be used if a sequencer is not available.
This protocol has been used routinely in our differential display projects, which involve more than 100 differential bands. By comparing with sequencing results using cloned cDNAs as templates, we have found that this method works well for nearly all the differential cDNA fragments longer than 150 bp. For obvious reasons, this procedure would not generate accurate sequences for cDNAs shorter than 100 bp. Fortunately, this is not a major problem. For northern-blot
110
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Technical Tips
analysis it is generally recommended to isolate differential bands longer than 250 bp (Ref. 1, 4). As mentioned in the protocol, the sizes of differential bands suitable for this procedure should optimally be longer than 200 bp. A large number of differential bands can be quickly characterized and sequenced using this procedure. The next step is to search a database in order to find if the differential sequences match any known genes or motifs. Meanwhile, northern-blot analysis should be performed to confirm the selective expression of the cDNA molecules. Based on these results, one could select a genuine candidate for further functional study.
References 1 Liang, P. and Pardee, A.B. (1992) Science 257, 967–971. 2 Wan, J.S. et al. (1996) Nat. Biotechnol. 14, 1685–1691. 3 Callard, D., Lescure, B. and Mazzolini, L. (1994) BioTechniques 16, 1096–1097. 4 Liang, P. et al. (1994) Nucleic Acids Res. 22, 5763–5764. 5 Li, F., Barnathan, E.S. and Kariko, K. (1994) Nucleic Acids Res. 22, 1764–1765. 6 Orita, M. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2766–2770. 7 Geisinger, A., Rodriguez, R., Romero, V. and Wettstein, R. (1997) Technical Tips Online. (http://www.elsevier.com/locate/tto). 8 Zhao, S., Ooi, S.L., Yang, F.C. and Pardee, A.B. (1996) BioTechniques 20, 400–404. 9 Poirier, G.M.C., Pyati, J., Wan, J.S. and Erlander, M.G. (1997) Nucleic Acids Res. 25, 913–914. 10 Corton, J.C. and Gustafsson, J.A. (1997) BioTechniques 22, 802–804. 11 Wawer, C., Ruggeberg, H., Meyer, G. and Muyzer, G. (1995) Nucleic Acids Res. 23, 4928–4929. 12 Issacs, S.T. et al. (1991) Nucleic Acids Res. 19, 109–116. 13 Linskens, M.H. et al. (1995) Nucleic Acids Res. 23, 3244–3251.
Products Used RNAimage kit: RNAimage kit from GenHunter Corporation Resolver Gold: Resolver Gold from R & D Systems Exonuclease I: Exonuclease I from Amersham Pharmacia Biotech Alkaline Phosphatase: Alkaline Phosphatase from Boehringer Mannheim alkaline phosphatase: alkaline phosphatase from Tropix Inc ABI Mix: ABI Mix from PE Applied Biosystems 377 sequencer: 377 sequencer from PE Applied Biosystems
Direct characterization, enrichment and sequencing of differential displayed cDNA sequences Jin Zenga,b, Mingfang Liua and Jean-Charles Frucharta a b
Institute Pasteur de Lille, 1 Rue Calmette, 59019 Lille, France NIMH/NIH, Building 10, Room 3N218, Bethesda, MD 20892, USA
Keywords: Polymerase chain reaction, Cloning & sequencing ▼Differential display of mRNA by PCR (DD-PCR) is a popular technique for cloning selectively expressed genes (Ref. 1). This method has some unique advantages over classical differential cloning methods (Ref. 2), such as the possibility for simultaneous comparison of mRNA differences among several samples. Its principle is elegantly simple; nevertheless, there are several practical difficulties with this technique. Most notably, owing to the fact that a significant number of differential fragments displayed as a single band on sequencing gels are heterogeneous (Ref. 3, 4, 5), true positive cDNAs must be differentiated from contaminating sequences by selection. Several methods for the postdisplay screening have been reported (Ref. 6, 7); however, their main utility is limited to identify distinct inserts from cloning. A complete post-display analysis often involves screening hundreds of clones for a handful of distinct and desirable cDNAs. It is a major bottleneck of differential display and has been recognized as among the most cumbersome steps of the method (Ref. 8, 9). Furthermore, it can be prohibitively expensive (Ref. 10). Here we present a rapid and cost-effective approach without recourse to cloning for direct characterization of differential cDNA molecules, up to the point of sequence identification. The experimental conditions have been optimized so that the protocol could be reliably performed with only three steps of minimal manipulation. It has the following novel features and advantages. First, our method of direct separation is very economical and effective. Like Geisinger et al. (Ref. 7, 11), we also use
the DNA ligand bisbenzimide to separate co-migrating sequences. However, our method does not involve cloning. As expected from the fact that differential cDNA sequences are mainly derived from AT-rich 3 -UTR regions of mRNA molecules to which bisbenzimide preferentially binds, major components of a vast majority of reamplified differential molecules could be directly separated by the DNA dye using the electrophoresis conditions stated below (data not shown). Second, cDNA fragments resolved by bisbenzimide can be directly enriched by PCR without prior purification. We observed that, by contrast with other DNA ligands such as isopsoralen (Ref. 12), bisbenzimide could not photochemically block amplification by Taq polymerase under the conditions (data not shown and see the protocol). Thus, no labor-intensive manipulation for cleaning up the PCR products is needed when using the procedure. Third, our method of separation and enrichment is highly efficient. The differential cDNA fragments recovered as such are usually so pure that they could even be reliably used in automatic sequencing under the optimized cycle reaction condition presented below.
Protocol 1.
2. Corresponding author: [email protected]
c 1366-2120 1998 Elsevier Ltd. All rights reserved. PII: S1366-2120(08)70115-6
Carry out differential display and reamplify differential bands (their sizes should optimally be more than 200 bp) after excising them from gel using the protocols of Linskens et al. (Ref. 13) or the secondgeneration RNAimage kit (GenHunter). Prepare a 2% agarose gel with bisbenzimide-PEG 6000 [‘Resolver GoldTM ’, (R&D System)] by adding 1 unit of
www.sciencedirect.com
109
Technical Tips Online, Vol. 3, 1998
3. 4. 5. 6. 7. 8.
9.
10.
11.
the agent per ml into agarose gel precooled to 70◦ C or less. Use 1×TAE, pH7.2 as gel and electrophoresis buffers (note that pH of the buffer is critical!). Load 20 µl of the reamplified differential cDNA samples with bromophenol blue. Run the electrophoresis at a voltage of 5 Vcm−1 until the dye reaches about two third of the gel. Stain the gel with 0.5 µg/ml of ethidium bromide. Visualize the bands using a UV lamp and remove them into an Eppendorf tube containing 100 µl of H2 O. Heat the gel band at 100◦ C for 5 min. Use 2 µl of the gel solution for PCR amplification: 4 µl 10×PCR buffer, 4 µl 25 mM MgCl2 , 3.2 µl 0.25 mM dNTP, 1.6 µl 10 mM 5 and 3 primers, 0.4 µl Taq polymerase and water to 40 µl. Carry out 30 cycles (94◦ C for 50 s; 60◦ C for 60 s; and 72◦ C for 60 s) with a final extension at 72◦ C for 10 min. Upon amplification, load 10 µl of the product onto a normal agarose gel for verification, if necessary. Pretreat the PCR amplified samples for sequencing: add 0.5 µl (10 U/µl) Exonuclease I (Amersham) and 0.5 µl (2 U/µl) Shrimp alkaline phosphatase (Amersham) into 2.5 µl of the re-amplified product in a PCR tube; incubate the reaction mixture at 37◦ C for 30 min, and then at 80◦ C for 15 min. Carry out the cycle sequencing reaction: add 0.5 µl 10 µM 5 random primer, 8µl ABI Mix (Perkin-Elmer), 8 µl H2 O directly into the pretreated reaction mixture. Carry out 25 cycles with an initial incubation at 96◦ C for 10 s (96◦ C, 10 s; 56◦ C, 5 s; 60◦ C, 240 s). Precipitate the sequencing reaction by ethanol as recommended by the manufacturer (Perkin-Elmer) and analyze the sequence using an ABI prism 377 sequencer (Perkin-Elmer). Note that manual sequencing could also be used if a sequencer is not available.
This protocol has been used routinely in our differential display projects, which involve more than 100 differential bands. By comparing with sequencing results using cloned cDNAs as templates, we have found that this method works well for nearly all the differential cDNA fragments longer than 150 bp. For obvious reasons, this procedure would not generate accurate sequences for cDNAs shorter than 100 bp. Fortunately, this is not a major problem. For northern-blot
110
www.sciencedirect.com
Technical Tips
analysis it is generally recommended to isolate differential bands longer than 250 bp (Ref. 1, 4). As mentioned in the protocol, the sizes of differential bands suitable for this procedure should optimally be longer than 200 bp. A large number of differential bands can be quickly characterized and sequenced using this procedure. The next step is to search a database in order to find if the differential sequences match any known genes or motifs. Meanwhile, northern-blot analysis should be performed to confirm the selective expression of the cDNA molecules. Based on these results, one could select a genuine candidate for further functional study.
References 1 Liang, P. and Pardee, A.B. (1992) Science 257, 967–971. 2 Wan, J.S. et al. (1996) Nat. Biotechnol. 14, 1685–1691. 3 Callard, D., Lescure, B. and Mazzolini, L. (1994) BioTechniques 16, 1096–1097. 4 Liang, P. et al. (1994) Nucleic Acids Res. 22, 5763–5764. 5 Li, F., Barnathan, E.S. and Kariko, K. (1994) Nucleic Acids Res. 22, 1764–1765. 6 Orita, M. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2766–2770. 7 Geisinger, A., Rodriguez, R., Romero, V. and Wettstein, R. (1997) Technical Tips Online. (http://www.elsevier.com/locate/tto). 8 Zhao, S., Ooi, S.L., Yang, F.C. and Pardee, A.B. (1996) BioTechniques 20, 400–404. 9 Poirier, G.M.C., Pyati, J., Wan, J.S. and Erlander, M.G. (1997) Nucleic Acids Res. 25, 913–914. 10 Corton, J.C. and Gustafsson, J.A. (1997) BioTechniques 22, 802–804. 11 Wawer, C., Ruggeberg, H., Meyer, G. and Muyzer, G. (1995) Nucleic Acids Res. 23, 4928–4929. 12 Issacs, S.T. et al. (1991) Nucleic Acids Res. 19, 109–116. 13 Linskens, M.H. et al. (1995) Nucleic Acids Res. 23, 3244–3251.
Products Used RNAimage kit: RNAimage kit from GenHunter Corporation Resolver Gold: Resolver Gold from R & D Systems Exonuclease I: Exonuclease I from Amersham Pharmacia Biotech Alkaline Phosphatase: Alkaline Phosphatase from Boehringer Mannheim alkaline phosphatase: alkaline phosphatase from Tropix Inc ABI Mix: ABI Mix from PE Applied Biosystems 377 sequencer: 377 sequencer from PE Applied Biosystems