[32]
SYNTHESIS OF c D N A s FROM SYNTHETIC OLIGONUCLEOTIDES
389
stained samples in the electron microscope. LMM forms paracrystals with characteristic repeats (143A and 430 ~,). General Conclusions from Expression of Myosin and Actin in Escherichia coli 1. Internal initiation of translation can result in vastly higher synthesis of a truncated protein than when the first methionine is used. We have observed this phenomenon for actin and myosin sequences. We have observed both internal initiation of transcription and translation from some myosin head sequences. Prior to expression the sequences should be scanned for a consensus Shine-Dalgarno sequence located 8 - 12 nucleotides upstream of an in-frame methionine. 2. Longer myosin head and rod sequences (454 amino acids) seem to be much more susceptible to proteolysis than shorter segments. Constitutive expression results in higher degrees of proteolysis than inducible expression. 3. Co-expression of MLC1 with the construct encoding amino acids 1 - 8 3 2 does not alter the solubility or degradation of the S 1 head. 4. Specific lysis conditions have been developed which yield largely soluble, nondenatured actin after expression in E. coll. Acknowledgments We thank Art Rovner and Marcela Bravo-Zehnder for communicating methods and A. Szent-Gyorgyi for help with the L M M purification scheme. This work was supported by NIH
Grant GM29090 to L.L.E.M. is a trainee of the Medical Scientist Training Program (T326GM7288). R.S. is supportedby a March of Dimes PredoctoralFellowship(18-88-30). S.F. is supportedby National ResearchServiceAward 5T32 GM07128.
[32] Synthesis of cDNAs from Synthetic Oligonucleotides Using Troponin C as an Example
By GONG-QIAOXu and SARAH E. HITCHCOCK-DEGREGORI Expression of recombinant proteins in Escherichia coli or eukaryotic cells has become a valuable tool of biochemists, cell biologists, and molecular biologists. While the most c o m m o n approach has been to isolate a cDNA from a library, there are instances in which gene synthesis from synthetic oligonucleotides is a feasible, if not economically preferable, METHODS IN ENZYMOLOGY,VOL. 196
Copyright© 1991by AcademicPress,Inc. All fightsof ~productionin any formreserved.
390
MOLECULAR AND GENETIC APPROACHES
[32]
alternative. For example, an investigator may know the amino acid sequence of the protein, but a cDNA has not been isolated or is not conveniently available. A cDNA for a protein of known sequence may be incomplete. In this case, a full-length sequence can be generated by gene synthesis without isolating the full length of cDNA from a library. Finally, an investigator may wish to design a peptide or protein longer than can be made using synthetic peptides or insert a designed peptide in a known protein as a cassette. In each of these cases, gene synthesis may be the most economical method, considering money and time, to obtain the desired DNA. Additional advantages are the opportunities to include desired restriction enzyme sites in the sequence and remove undesired ones, important considerations if there are plans for site-directed mutagenesis. It is also possible to select preferred codons for the organism to be used for overexpression (bacteria, yeast, or higher eukaryotes), though the general importance of this for efficient expression is not well established. While synthetic cDNAs offer advantages for DNA manipulation in protein design, a limitation is that they are not suitable for studies of gene structure or expression or in vivo manipulations at the genomic or mRNA levels, since the DNA sequence is not that of the naturally occurring gene. In the cytoskeleton and cell motility field, gene synthesis has been used successfully for construction of cDNAs for calmodulin z and troponin C. 2 The present chapter describes a method for cDNA synthesis from synthetic oligonucleotides that was used for synthesis of a troponin C cDNA. 2 The investigator will need to become familiar with commonly used methods in recombinant DNA technology described in detail in other volumes in this series3 and in a number of excellent methods manuals. In addition, design of the DNA and oligonucleotides requires extensive sequence analysis and manipulation using commercially available programs for a personal or mainframe computer. DNA* programs (DNASTAR, Inc., Madison, WI) were used for the troponin C cDNA design. Design of cDNA Sequence There are two basic approaches to gene synthesis: full two-strand synthesis and synthesis by the overlapping fill-in method. The full two-strand synthesis is the safer, more widely used approach and was used for the i D. M. Roberts, R. Crea, M. Malecha, G. Alvarado-Urbina, R. H. Chiarello, and D. M. Watterson, Biochemistry 24, 5090 (1985). 2 G.-Q. Xu, and S. E. Hiteheock-DeGregori, J. Biol. Chem. 263, 13962 (1988). 3 This series, Vols. 68, 100, 101,152, 153, 154, and 155.
[32]
SYNTHESISOF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
391
calmodulin cDNA synthesis? Oligonucleotides for both coding and noncoding strands are synthesized. Overlapping complementary oligonucleotides are phosphorylated at the 5' ends, annealed, and the ends, overlapping by 6 - 8 bases, are joined by enzymatic ligation. The overlapping fill-in method 4,5 requires oligonucleotides that overlap at their 3' ends. The length of the overlap region depends on the temperature of the synthesis reaction and the GC content in this region. The double-stranded DNA is completed by enzymatic DNA synthesis from the 3' to the 5' end using the complementary strand as a template (Fig. 1). The major advantage is economic; the cost may be about 60% of a complete two-strand synthesis, depending on the column and per-base charges for the oligonucleotides. The disadvantages are the risk of mutation during enzymatic DNA synthesis and possible problems due to oligonucleotide secondary structure. Both approaches require careful sequence analysis of the completed gene. Difficulty due to secondary structure is hard to predict, but it may be minimized by careful oligonucleotide design. The overlapping fill-in method used for the troponin C cDNA synthesis2 is described in the present chapter. However, many of the considerations discussed apply to both methods. Successful gene synthesis and full benefit of the advantages offered by the approach require careful attention to cDNA and oligonucleotide design. 1. Using a computer program (for example, the DNA* program REVTRANS), reverse translate the protein sequence, including an initial methionine if appropriate, into DNA with the desired codon usage. 2. Add two or more stop codons at the 3' end of the cDNA if the DNA will be used for recombinant protein synthesis. 3. If the gene is to be assembled in segments (see below), search the DNA sequence for unique restriction sites that will be convenient for assembling the synthetic DNA segments into the full-length gene. Introduce, or remove, sites as appropriate, without changing the amino acid sequence. This can usually be done by changing the third base in a codon to introduce the desired restriction site without changing the amino acid sequence of the protein. In the case of the troponin C cDNA synthesis, of the two restriction sites used for assembly of the gene, the H i n c l I site was in the original reverse-translated sequence while the ClaI site was introduced by changing a C to a T (Fig. 1.). 4j. j. Rossi, R. Kierzek,T. Huang, P. A. Walker,and K. Itakura,J. Biol. Chem. 257, 9226 (1982). 5S. P. Adamsand G. R. Gallupi, Med. Res. Rev. 6, 135 (1986).
392
[32]
MOLECULAR AND GENETIC APPROACHES
Full cDNA Clai
Hincn
5' AT~
I
stop 3' codons
I
SeQment A 5' gcggatccATGG
cttaagcg5' HincIi EcoRI
BamH] NcoI
SeqmentB 5' gcggatcc
cttaagcg5' ClaI EcoRI
BamH] /-linch
SeqmentC 5' gcggatcc BamI~ ClaI
3' 3'
ATTATCttaagttcgaacg5' stop EcoPJ HindllI codons
Fie. 1. Synthesisof a troponinC eDNA from segments.
4. Search the DNA sequence for internal restriction enzyme sites that may be used later for cloning. For example, sites commonly used for cloning, such as EcoRI, HindIII, BamHI, NcoI, and other sites used in vector polylinkers should be removed as appropriate. Remove the sites without changing the amino acid sequence of the protein. 5. Search the DNA sequence for unique restriction sites that may be useful for subsequent cassette mutagenesis. Alter the DNA sequence to introduce desired sites and remove undesired ones. 6. After all the changes have been made, check the final restriction map of the eDNA and the amino acid sequence of the product.
Design of Oligonucleotides Oligonucleotides of 100 or more bases can generally be made without difficulty. The oligonucleofides used for the troponin C eDNA synthesis were 92-124 bases. If the final DNA is more than 200 bases long, the investigator will have to decide if it is to be assembled from segments, as described below, or if the synthesis of the entire eDNA will be done in one step from a mixture of several oligonucleotides. Assembly from segments is safer, but requires more work and is slightly more expensive in terms of oligonucleotide synthesis. Synthesis of the gene in segments may offer advantages for sequence determination, cassette mutagencsis, or exprcs-
[32]
SYNTHESISOF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
393
sion of a fragment of the protein. Genes have been synthesized successfully from mixtures of several oligonucleotides. However, if there is a "problem" oligonucleotide, the entire synthesis may fail. 1. Select sections of about 150-200 base pairs in the final gene, determined previously if the gene is to be assembled in segments (see Design of cDNA Sequence, step 3, above). 2. Add restriction enzyme sites at both ends of each section for use in subcloning the segments and the final gene assembly. The selection of cloning sites will depend on the vectors used and the strategy of the gene design. In the example shown in Fig. 1, an NcoI site was completed at the initiator ATG and the G of the codon of the next amino acid, alanine, to allow cloning in expression vectors. The HinclI and ClaI sites used to assemble the three segments were completed at the appropriate ends of the segments to allow joining of segment A to B and segment B to C (Fig. 2). BamHI and EcoRI sites for cloning were added at the beginning and end, respectively, of each segment. A HindIII site for cloning was added at the 3' end of the last segment. If the gene will be assembled from several oligonucleotides pairs, rather than from subcloned segments, restriction sites will be needed only at the 5' and 3' ends of the final gene. However, regions of overlap at the 5' end of the oligonucleotides, as well as the 3' ends, will need to be designed. 3. To create oligomer pairs for synthesis of each segment using the overlapping fill-in method, first set the temperature for carrying out the DNA polymerase reaction. The Klenow fragment ofE. coli DNA polymerase I was used at 46.5 ° for the troponin C cDNA synthesis. The higher reaction temperature decreases nonspecific annealing between two oligomers and reduces the secondary structure of each oligomer. Then choose a region around the middle of each section for the 3' overlap between the two oligonucleotides (Fig. 1). One oligonucleotide will encode the coding strand, the other the noncoding strand, and they will overlap at their 3' ends. The dissociation temperature (Td) of the overlap region can be estimated using an empirically determined formula by counting the number of AT and GC pairs (2°/AT pair, 4°/GC pairr'7). It should be at or above the temperature used for the DNA synthesis. The oligonucleotide pairs used for the troponin C synthesis had overlaps of 15 - 17 oligonucleotides with T d values of 46-48 o, close to the synthesis reaction temperature (46.5 ° ), thereby allowing stable annealing of the 3' overlap region. When6S. V. Suggs,T. Hirose, T. Miyaki,E. H. Kawashimi,M. J. Johnson, K. Itakura, and R. B. Wallace, ICN- UCLA Symp. Dev. BioL 23, 683 (1981). 7G. M. Wahl, S. L. Berger,and A. R. Kimmel,this series,Vol. 152,p. 399.
394
MOLECULARAND GENETICAPPROACHES
[32]
ever possible, the first and last residues in the overlaps should be GC pairs. Try to avoid the use of A at the 3' end since A may depurinate during oligonucleotide synthesis. 4. Analyze the oligonucleotide sequences for G content and presence of runs of G. There are reports of guanine modifications during solid-phase phosphoramidite synthesis that may lead to chain cleavage or mutagenesis. 8,9 Attempt to keep runs of G to three or less, since longer ones may aggregate or form other structures. In the troponin C oligonucleotides, no attempt was made to reduce the overall G content. There were no places with more than three consecutive G's in the original reverse-translated sequence. 5. After designing the oligonucleotide pairs, analyze the sequences for regions of pairing other than at the overlap region. To do this, use a computer program (for example, the DNA* program COMPARE) to search for homology between one oligonucleotide and the complementary sequence of its pair (generated using the DNA* program INREV). The oligonucleotides should also be evaluated for formation of secondary structure that may interfere with DNA synthesis. To do this, use a program (for example, the DNA* program LOOPS) to search for dyad symmetries that would allow formation of loop stem structures within an individual oligomer. Compare the Td values of undesirable homologous and stem regions with the synthesis reaction temperature. If there are homologous or stem regions with Td values close to the reaction temperature, change the sequence accordingly. The longest undesirable region in the troponin C oligonucleotides was 7 base pairs with a calculated Td of 22 °, not of concern since it was well below that of the overlap region and the synthesis reaction temperature. 6. If any sequence changes were made in steps 3-5, reassemble the troponin C cDNA sequence (in the computer) and check it for the desired restriction sites and amino acid sequence of the product. 7. Synthesize the oligonucleotides, purify them chromatographically or electrophoretically. Generally this is done by an in-house or commercial DNA synthesis facility. Analyze the purified oligonucleotides for purity electrophoretically or using high-pressure liquid chromatography (HPLC). Synthesis of Segments and Assembly of Complete cDNA The last stage of DNA synthesis and cloning employs methods that are now conventional to recombinant DNA technology and therefore will be described in only minimal detail. The procedure is straight forward. 8 R. T. Pon, M. J. Damha, and K. K. Ogiivie, Nucleic Acids Res. 13, 6447 (1985). 9 j. S. Eadie and D. S. Davidson, NucleicAeids Res. 15, 8333 (1987).
[32]
SYNTHESIS OF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
395
1. To fall in the overlapping oligonucleotides, DNA polymerization is carried out using the Klenow fragment of E. coli DNA polymerase I. To anneal the overlapping oligonucleotide pairs, incubate 40 pmol of each of the two oligonucleotides in a pair in 34/zl of 8 m M Tris-HC1, pH 7.5, 8 m M MgCI2, 60 m M NaC1 in a boiling water bath for 3 min, transfer to 56 ° for 5 min, and finally to 46.5 ° for 30 min. For DNA polymerization, add 4/zl of a solution of the four deoxyribonucleotide triphosphates (500/zM) and 2/zl of Klenow fragment (1 unit//zl) to the annealed DNA, making a final volume of 40/d. Incubate for another 20 min at 46.5 °. Although there is sufficient product in a reaction to visualize an aliquot on an agarose or polyacrylamide gel after ethidium bromide staining, labeled dNTPs may be added during synthesis to allow autoradiographic analysis of the product. Alternatively, the oligonucleotide may be phosphorylated using [~2P]ATP as a substrate. Terminate the reaction by extracting once with phenol/chloroform and twice with chloroform. Add sodium acetate, pH 5.2, to 0.3 M, and precipitate the DNA by addition of 2 vol of ethanol, centrifuge, rinse once with 70% ethanol, and dry. Dissolve the dried pellet containing the synthesized fragment in H20 or TE buffer (10 m M Tris HC1, pH 7.5-8; 1 m M EDTA) and digest the DNA with the appropriate restriction endonucleases (BamHI and EcoRI or HindlII in the case of the troponin C example). Following phenol/chloroform and chloroform extraction and ethanol precipitation as before, dissolve the dried pellet in 10 #1 H20 for electrophoretic analysis and cloning. Complete synthesis is calculated to yield about 4/zg DNA of a 200 mer. 2. Electrophoretic analysis of the synthesized DNA should show a clear band of the expected length visible with ethidium bromide. The type of gel used will depend on the expected molecular weight of the product. Higher molecular weight products may be present due the tendency of E. coli DNA polymerase I to copy the newly synthesized strand at higher temperatures (20 ° and higher), producing "snapback" DNA. ~° While considerable higher molecular weight material was seen in the synthesized troponin C segments (see Fig. 2, Ref. 2), it did not interfere with the subsequent cloning steps. 3. Clone the individual segments in an appropriate vector (1 - 2 #1 in a volume of 20/zl), prepare plasmid or M 13 RF DNA from recombinants, digest it with the appropriate restriction enzymes, and compare the length of the insert with that of the synthesized DNA on a gel. Isolate the segment from the gel, ligate it to the next segment, and subclone it again. Continue until the complete cDNA has been assembled. In the case of troponin C, it was a two-step process (Fig. 2). t0 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
396
[32]
MOLECULAR A N D GENETIC APPROACHES
4. Determine the complete nucleotide sequence following each subcloning step in the assembly and after the complete cDNA is assembled.
HinclI
BamHI
~BamHI
Ligation HincN
BamHI
+ Hindl
ClaI
~
BamHI
BamHI+ ClaI ~ C l a I
+ Hiret[l]
IN Ligatio n
~ ClaI
BamHI +
ClaI
Hinc]l
FI6.2. Scheme for assembly ofa troponin C cDNA. The initial segments (A, B, C; see Fig. 1) were cloned in M13 mpl8 or 19. Segments were isolated electrophoretically from agarose following digestion with the indicated enzymes. Segment B (BamHl, Clal was ligated to Segment C (Clal, HindlII digest) and cloned in M 13 mpl9 at the BamHI and HindlII sites. To assemble the complete cDNA, segment A (BamHI, HinclI digest) was ligated to segment BC (HinclI, HindllI digest) and cloned in M13 mpl8 or 19 at the BamHI and HindlII sites. The complete cDNA was then isolated (NcoI, EcoRI, or HindlII digest) for cloning in expression vectors.
SYNTHESIS OF c D N A s FROM SYNTHETIC OLIGONUCLEOTIDES
389
stained samples in the electron microscope. LMM forms paracrystals with characteristic repeats (143A and 430 ~,). General Conclusions from Expression of Myosin and Actin in Escherichia coli 1. Internal initiation of translation can result in vastly higher synthesis of a truncated protein than when the first methionine is used. We have observed this phenomenon for actin and myosin sequences. We have observed both internal initiation of transcription and translation from some myosin head sequences. Prior to expression the sequences should be scanned for a consensus Shine-Dalgarno sequence located 8 - 12 nucleotides upstream of an in-frame methionine. 2. Longer myosin head and rod sequences (454 amino acids) seem to be much more susceptible to proteolysis than shorter segments. Constitutive expression results in higher degrees of proteolysis than inducible expression. 3. Co-expression of MLC1 with the construct encoding amino acids 1 - 8 3 2 does not alter the solubility or degradation of the S 1 head. 4. Specific lysis conditions have been developed which yield largely soluble, nondenatured actin after expression in E. coll. Acknowledgments We thank Art Rovner and Marcela Bravo-Zehnder for communicating methods and A. Szent-Gyorgyi for help with the L M M purification scheme. This work was supported by NIH
Grant GM29090 to L.L.E.M. is a trainee of the Medical Scientist Training Program (T326GM7288). R.S. is supportedby a March of Dimes PredoctoralFellowship(18-88-30). S.F. is supportedby National ResearchServiceAward 5T32 GM07128.
[32] Synthesis of cDNAs from Synthetic Oligonucleotides Using Troponin C as an Example
By GONG-QIAOXu and SARAH E. HITCHCOCK-DEGREGORI Expression of recombinant proteins in Escherichia coli or eukaryotic cells has become a valuable tool of biochemists, cell biologists, and molecular biologists. While the most c o m m o n approach has been to isolate a cDNA from a library, there are instances in which gene synthesis from synthetic oligonucleotides is a feasible, if not economically preferable, METHODS IN ENZYMOLOGY,VOL. 196
Copyright© 1991by AcademicPress,Inc. All fightsof ~productionin any formreserved.
390
MOLECULAR AND GENETIC APPROACHES
[32]
alternative. For example, an investigator may know the amino acid sequence of the protein, but a cDNA has not been isolated or is not conveniently available. A cDNA for a protein of known sequence may be incomplete. In this case, a full-length sequence can be generated by gene synthesis without isolating the full length of cDNA from a library. Finally, an investigator may wish to design a peptide or protein longer than can be made using synthetic peptides or insert a designed peptide in a known protein as a cassette. In each of these cases, gene synthesis may be the most economical method, considering money and time, to obtain the desired DNA. Additional advantages are the opportunities to include desired restriction enzyme sites in the sequence and remove undesired ones, important considerations if there are plans for site-directed mutagenesis. It is also possible to select preferred codons for the organism to be used for overexpression (bacteria, yeast, or higher eukaryotes), though the general importance of this for efficient expression is not well established. While synthetic cDNAs offer advantages for DNA manipulation in protein design, a limitation is that they are not suitable for studies of gene structure or expression or in vivo manipulations at the genomic or mRNA levels, since the DNA sequence is not that of the naturally occurring gene. In the cytoskeleton and cell motility field, gene synthesis has been used successfully for construction of cDNAs for calmodulin z and troponin C. 2 The present chapter describes a method for cDNA synthesis from synthetic oligonucleotides that was used for synthesis of a troponin C cDNA. 2 The investigator will need to become familiar with commonly used methods in recombinant DNA technology described in detail in other volumes in this series3 and in a number of excellent methods manuals. In addition, design of the DNA and oligonucleotides requires extensive sequence analysis and manipulation using commercially available programs for a personal or mainframe computer. DNA* programs (DNASTAR, Inc., Madison, WI) were used for the troponin C cDNA design. Design of cDNA Sequence There are two basic approaches to gene synthesis: full two-strand synthesis and synthesis by the overlapping fill-in method. The full two-strand synthesis is the safer, more widely used approach and was used for the i D. M. Roberts, R. Crea, M. Malecha, G. Alvarado-Urbina, R. H. Chiarello, and D. M. Watterson, Biochemistry 24, 5090 (1985). 2 G.-Q. Xu, and S. E. Hiteheock-DeGregori, J. Biol. Chem. 263, 13962 (1988). 3 This series, Vols. 68, 100, 101,152, 153, 154, and 155.
[32]
SYNTHESISOF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
391
calmodulin cDNA synthesis? Oligonucleotides for both coding and noncoding strands are synthesized. Overlapping complementary oligonucleotides are phosphorylated at the 5' ends, annealed, and the ends, overlapping by 6 - 8 bases, are joined by enzymatic ligation. The overlapping fill-in method 4,5 requires oligonucleotides that overlap at their 3' ends. The length of the overlap region depends on the temperature of the synthesis reaction and the GC content in this region. The double-stranded DNA is completed by enzymatic DNA synthesis from the 3' to the 5' end using the complementary strand as a template (Fig. 1). The major advantage is economic; the cost may be about 60% of a complete two-strand synthesis, depending on the column and per-base charges for the oligonucleotides. The disadvantages are the risk of mutation during enzymatic DNA synthesis and possible problems due to oligonucleotide secondary structure. Both approaches require careful sequence analysis of the completed gene. Difficulty due to secondary structure is hard to predict, but it may be minimized by careful oligonucleotide design. The overlapping fill-in method used for the troponin C cDNA synthesis2 is described in the present chapter. However, many of the considerations discussed apply to both methods. Successful gene synthesis and full benefit of the advantages offered by the approach require careful attention to cDNA and oligonucleotide design. 1. Using a computer program (for example, the DNA* program REVTRANS), reverse translate the protein sequence, including an initial methionine if appropriate, into DNA with the desired codon usage. 2. Add two or more stop codons at the 3' end of the cDNA if the DNA will be used for recombinant protein synthesis. 3. If the gene is to be assembled in segments (see below), search the DNA sequence for unique restriction sites that will be convenient for assembling the synthetic DNA segments into the full-length gene. Introduce, or remove, sites as appropriate, without changing the amino acid sequence. This can usually be done by changing the third base in a codon to introduce the desired restriction site without changing the amino acid sequence of the protein. In the case of the troponin C cDNA synthesis, of the two restriction sites used for assembly of the gene, the H i n c l I site was in the original reverse-translated sequence while the ClaI site was introduced by changing a C to a T (Fig. 1.). 4j. j. Rossi, R. Kierzek,T. Huang, P. A. Walker,and K. Itakura,J. Biol. Chem. 257, 9226 (1982). 5S. P. Adamsand G. R. Gallupi, Med. Res. Rev. 6, 135 (1986).
392
[32]
MOLECULAR AND GENETIC APPROACHES
Full cDNA Clai
Hincn
5' AT~
I
stop 3' codons
I
SeQment A 5' gcggatccATGG
cttaagcg5' HincIi EcoRI
BamH] NcoI
SeqmentB 5' gcggatcc
cttaagcg5' ClaI EcoRI
BamH] /-linch
SeqmentC 5' gcggatcc BamI~ ClaI
3' 3'
ATTATCttaagttcgaacg5' stop EcoPJ HindllI codons
Fie. 1. Synthesisof a troponinC eDNA from segments.
4. Search the DNA sequence for internal restriction enzyme sites that may be used later for cloning. For example, sites commonly used for cloning, such as EcoRI, HindIII, BamHI, NcoI, and other sites used in vector polylinkers should be removed as appropriate. Remove the sites without changing the amino acid sequence of the protein. 5. Search the DNA sequence for unique restriction sites that may be useful for subsequent cassette mutagenesis. Alter the DNA sequence to introduce desired sites and remove undesired ones. 6. After all the changes have been made, check the final restriction map of the eDNA and the amino acid sequence of the product.
Design of Oligonucleotides Oligonucleotides of 100 or more bases can generally be made without difficulty. The oligonucleofides used for the troponin C eDNA synthesis were 92-124 bases. If the final DNA is more than 200 bases long, the investigator will have to decide if it is to be assembled from segments, as described below, or if the synthesis of the entire eDNA will be done in one step from a mixture of several oligonucleotides. Assembly from segments is safer, but requires more work and is slightly more expensive in terms of oligonucleotide synthesis. Synthesis of the gene in segments may offer advantages for sequence determination, cassette mutagencsis, or exprcs-
[32]
SYNTHESISOF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
393
sion of a fragment of the protein. Genes have been synthesized successfully from mixtures of several oligonucleotides. However, if there is a "problem" oligonucleotide, the entire synthesis may fail. 1. Select sections of about 150-200 base pairs in the final gene, determined previously if the gene is to be assembled in segments (see Design of cDNA Sequence, step 3, above). 2. Add restriction enzyme sites at both ends of each section for use in subcloning the segments and the final gene assembly. The selection of cloning sites will depend on the vectors used and the strategy of the gene design. In the example shown in Fig. 1, an NcoI site was completed at the initiator ATG and the G of the codon of the next amino acid, alanine, to allow cloning in expression vectors. The HinclI and ClaI sites used to assemble the three segments were completed at the appropriate ends of the segments to allow joining of segment A to B and segment B to C (Fig. 2). BamHI and EcoRI sites for cloning were added at the beginning and end, respectively, of each segment. A HindIII site for cloning was added at the 3' end of the last segment. If the gene will be assembled from several oligonucleotides pairs, rather than from subcloned segments, restriction sites will be needed only at the 5' and 3' ends of the final gene. However, regions of overlap at the 5' end of the oligonucleotides, as well as the 3' ends, will need to be designed. 3. To create oligomer pairs for synthesis of each segment using the overlapping fill-in method, first set the temperature for carrying out the DNA polymerase reaction. The Klenow fragment ofE. coli DNA polymerase I was used at 46.5 ° for the troponin C cDNA synthesis. The higher reaction temperature decreases nonspecific annealing between two oligomers and reduces the secondary structure of each oligomer. Then choose a region around the middle of each section for the 3' overlap between the two oligonucleotides (Fig. 1). One oligonucleotide will encode the coding strand, the other the noncoding strand, and they will overlap at their 3' ends. The dissociation temperature (Td) of the overlap region can be estimated using an empirically determined formula by counting the number of AT and GC pairs (2°/AT pair, 4°/GC pairr'7). It should be at or above the temperature used for the DNA synthesis. The oligonucleotide pairs used for the troponin C synthesis had overlaps of 15 - 17 oligonucleotides with T d values of 46-48 o, close to the synthesis reaction temperature (46.5 ° ), thereby allowing stable annealing of the 3' overlap region. When6S. V. Suggs,T. Hirose, T. Miyaki,E. H. Kawashimi,M. J. Johnson, K. Itakura, and R. B. Wallace, ICN- UCLA Symp. Dev. BioL 23, 683 (1981). 7G. M. Wahl, S. L. Berger,and A. R. Kimmel,this series,Vol. 152,p. 399.
394
MOLECULARAND GENETICAPPROACHES
[32]
ever possible, the first and last residues in the overlaps should be GC pairs. Try to avoid the use of A at the 3' end since A may depurinate during oligonucleotide synthesis. 4. Analyze the oligonucleotide sequences for G content and presence of runs of G. There are reports of guanine modifications during solid-phase phosphoramidite synthesis that may lead to chain cleavage or mutagenesis. 8,9 Attempt to keep runs of G to three or less, since longer ones may aggregate or form other structures. In the troponin C oligonucleotides, no attempt was made to reduce the overall G content. There were no places with more than three consecutive G's in the original reverse-translated sequence. 5. After designing the oligonucleotide pairs, analyze the sequences for regions of pairing other than at the overlap region. To do this, use a computer program (for example, the DNA* program COMPARE) to search for homology between one oligonucleotide and the complementary sequence of its pair (generated using the DNA* program INREV). The oligonucleotides should also be evaluated for formation of secondary structure that may interfere with DNA synthesis. To do this, use a program (for example, the DNA* program LOOPS) to search for dyad symmetries that would allow formation of loop stem structures within an individual oligomer. Compare the Td values of undesirable homologous and stem regions with the synthesis reaction temperature. If there are homologous or stem regions with Td values close to the reaction temperature, change the sequence accordingly. The longest undesirable region in the troponin C oligonucleotides was 7 base pairs with a calculated Td of 22 °, not of concern since it was well below that of the overlap region and the synthesis reaction temperature. 6. If any sequence changes were made in steps 3-5, reassemble the troponin C cDNA sequence (in the computer) and check it for the desired restriction sites and amino acid sequence of the product. 7. Synthesize the oligonucleotides, purify them chromatographically or electrophoretically. Generally this is done by an in-house or commercial DNA synthesis facility. Analyze the purified oligonucleotides for purity electrophoretically or using high-pressure liquid chromatography (HPLC). Synthesis of Segments and Assembly of Complete cDNA The last stage of DNA synthesis and cloning employs methods that are now conventional to recombinant DNA technology and therefore will be described in only minimal detail. The procedure is straight forward. 8 R. T. Pon, M. J. Damha, and K. K. Ogiivie, Nucleic Acids Res. 13, 6447 (1985). 9 j. S. Eadie and D. S. Davidson, NucleicAeids Res. 15, 8333 (1987).
[32]
SYNTHESIS OF cDNAs FROMSYNTHETICOLIGONUCLEOTIDES
395
1. To fall in the overlapping oligonucleotides, DNA polymerization is carried out using the Klenow fragment of E. coli DNA polymerase I. To anneal the overlapping oligonucleotide pairs, incubate 40 pmol of each of the two oligonucleotides in a pair in 34/zl of 8 m M Tris-HC1, pH 7.5, 8 m M MgCI2, 60 m M NaC1 in a boiling water bath for 3 min, transfer to 56 ° for 5 min, and finally to 46.5 ° for 30 min. For DNA polymerization, add 4/zl of a solution of the four deoxyribonucleotide triphosphates (500/zM) and 2/zl of Klenow fragment (1 unit//zl) to the annealed DNA, making a final volume of 40/d. Incubate for another 20 min at 46.5 °. Although there is sufficient product in a reaction to visualize an aliquot on an agarose or polyacrylamide gel after ethidium bromide staining, labeled dNTPs may be added during synthesis to allow autoradiographic analysis of the product. Alternatively, the oligonucleotide may be phosphorylated using [~2P]ATP as a substrate. Terminate the reaction by extracting once with phenol/chloroform and twice with chloroform. Add sodium acetate, pH 5.2, to 0.3 M, and precipitate the DNA by addition of 2 vol of ethanol, centrifuge, rinse once with 70% ethanol, and dry. Dissolve the dried pellet containing the synthesized fragment in H20 or TE buffer (10 m M Tris HC1, pH 7.5-8; 1 m M EDTA) and digest the DNA with the appropriate restriction endonucleases (BamHI and EcoRI or HindlII in the case of the troponin C example). Following phenol/chloroform and chloroform extraction and ethanol precipitation as before, dissolve the dried pellet in 10 #1 H20 for electrophoretic analysis and cloning. Complete synthesis is calculated to yield about 4/zg DNA of a 200 mer. 2. Electrophoretic analysis of the synthesized DNA should show a clear band of the expected length visible with ethidium bromide. The type of gel used will depend on the expected molecular weight of the product. Higher molecular weight products may be present due the tendency of E. coli DNA polymerase I to copy the newly synthesized strand at higher temperatures (20 ° and higher), producing "snapback" DNA. ~° While considerable higher molecular weight material was seen in the synthesized troponin C segments (see Fig. 2, Ref. 2), it did not interfere with the subsequent cloning steps. 3. Clone the individual segments in an appropriate vector (1 - 2 #1 in a volume of 20/zl), prepare plasmid or M 13 RF DNA from recombinants, digest it with the appropriate restriction enzymes, and compare the length of the insert with that of the synthesized DNA on a gel. Isolate the segment from the gel, ligate it to the next segment, and subclone it again. Continue until the complete cDNA has been assembled. In the case of troponin C, it was a two-step process (Fig. 2). t0 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
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MOLECULAR A N D GENETIC APPROACHES
4. Determine the complete nucleotide sequence following each subcloning step in the assembly and after the complete cDNA is assembled.
HinclI
BamHI
~BamHI
Ligation HincN
BamHI
+ Hindl
ClaI
~
BamHI
BamHI+ ClaI ~ C l a I
+ Hiret[l]
IN Ligatio n
~ ClaI
BamHI +
ClaI
Hinc]l
FI6.2. Scheme for assembly ofa troponin C cDNA. The initial segments (A, B, C; see Fig. 1) were cloned in M13 mpl8 or 19. Segments were isolated electrophoretically from agarose following digestion with the indicated enzymes. Segment B (BamHl, Clal was ligated to Segment C (Clal, HindlII digest) and cloned in M 13 mpl9 at the BamHI and HindlII sites. To assemble the complete cDNA, segment A (BamHI, HinclI digest) was ligated to segment BC (HinclI, HindllI digest) and cloned in M13 mpl8 or 19 at the BamHI and HindlII sites. The complete cDNA was then isolated (NcoI, EcoRI, or HindlII digest) for cloning in expression vectors.
