[42]
PCR
CLONING OF ION CHANNEL GENE FAMILIES
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[42] C l o n i n g o f I o n C h a n n e l G e n e F a m i l i e s U s i n g t h e Polymerase Chain Reaction B y ELEAZARC. VE6A-SAENZ DE MIERA and JEN-WEI LIN
Introduction The polymerase chain reaction (PCR), developed by Mullis et al., ~ is a method to amplify a fragment of DNA sequence by using specific primers that flank a region of interest. Thermostable polymerases are used for repeated DNA synthesis from a given template. This method has been used in various stages of ion channel cloning.2-6 When properly used, it is an extremely powerful technique, and one may obtain more than 106-fold amplification. Many reviews on the theory and applications of PCR methods are availableT,8; interested readers may obtain basic information from these sources. We describe here the use of the PCR to identify putative members of an ion channel gene family. In comparison to low-stringency hybridization, we have found the PCR to be a faster and more efficient method to discover new members of the family. For this purpose, the template for the PCR can be either genomic DNA 9 or cDNA. The cDNA may be derived from a cDNA library ~° or, as in the example described here, may be single-stranded cDNA synthesized from the mRNA of a tissue of interest. In addition, if cDNA is used as template, the amplified
K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich, Cold Spring Harbor Syrup. Quant. Biol. 51, 263 (1986). 2 G. I. Fishman, D. C. Spray, and L. A. Leinwand, J. CellBiol. 111, 589 (1990). 3 K. Folander, J. S. Smith, J. Antanavage, C. Bennett, R. B. Stein, and R. Swanson, Proc. Natl. Acad. Sci. U.S.A. 87, 2975 (1990). 4 K. Otsu, H. F. Willard, V. K. Khanna, F. Zorzato, N. M. Green, and D. H. MacLennan, J. Biol. Chem. 265, 13472 (1990). 5 j. C. L. Tseng-Crank, G.-Y. Tseng, A. Schwartz, and M. A. Tanouye, FEBS Left. 268, 63 (1990). 6 Z-Y. Zhao and R. H. Joho, Biochem. Biophys. Res. Commun. 167, 174 (1990). 7 H. A. Erlich, ed., "PCR Technology: Principles and Applications for DNA Amplification." Stockton, New York, 1989. s M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White eds., "PCR Protocols: A Guide to Methods and Applications." Academic Press, San Diego, California, 1990. 9 A. Kamb, M. Weir, B. Rudy, H. Varmus, and C. Kenyon, Proc. Natl. Acad. Sci. U.S.A. 86, 4372 (1989). 1oB. F. O'Dowd, T. Nguyen, A. Tirpak, K. R. Jarvie, Y. Israel, P. Seeman, and H. B. Niznik, FEBS Lett. 262, 8 (1990).
METHODS IN ENZYMOLOGY, VOL. 207
Copyright © 1992 by Academic Press, Inc. All fights of reproduction in any form reserved.
614
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
PCR fragments provide a profile of gene expression in a specific tixsue.H In principle, this can be done with RNA from a very small amount of tissue. ~2 In this chapter, we discuss the choice and design of PCR primers. Detailed PCR protocols provided here have worked for us and are generally applicable in most circumstances. Design of Primers The choice and design of primers constitute perhaps the most important variable for a successful PCR. To optimize the identification of distinct members of a gene family, one should amplify a region of a gene that is variable within the family. This variable region should be flanked by conserved areas where the PCR primers will be located. Therefore, the design of primers depends on previous knowledge of the sequence of some member(s) of the family. For general considerations of primer design, good sources of references are available. 7.S Some special considerations for the amplification of members of a gene family are listed below. Selecting Regionsfor Amplification. The first step is to determine the regions of the channel to be amplified. One should start with collecting as many examples of existing amino acid sequences of members of a gene family as possible so that conserved regions can be identified by sequence comparison. In case that only one or a few members of the family are known, and doubts exist about the likelihood of conservation of certain regions, knowledge or assumptions of amino acid sequence domains with specific functional roles can be used as a guide. For example, based on the speculations that the H5 region of Shaker potassium channels is involved in the selectivity for potassium ions, 9 one may design a primer from this region. (A hybrid arrest experiment may be used to evaluate the validity of the assumptions; see [41] in this volume.) In a typical PCR two conserved regions are necessary so that the sequence flanked by two primers is amplified. However, it is not necessary that both primers hybridize to sequences in the channel eDNA. For example, to amplify from library eDNA, '° one may use a primer that hybridizes to the sequence of interest and a second primer that hybridizes to sequences on a host cloning vector. Minimizing Codon Degeneracy. After conserved areas are identified, the amino acid sequence of the regions should be analyzed to locate a stretch of amino acid sequence with minimal codon degeneracy. (PCR primers typically have a length of 18 to 30 nucleotides. 8) Given the asit E. Vega-Saenz de Micra, N. Chiu, K. Sen, D. Lau, J. W. Lin, and B. Rudy, Biophys. J. 59, 197a (1991). 12 H. H. Li, U. B. Gyllensten, X. F. Cui, R. K. Saiki, and H. A. Erfieh, Nature (London) 335, 414(1988).
[42]
P C R CLONING OF ION CHANNEL GENE FAMILIES
615
sumption that only amino acid sequence is conserved in the course of evolution, it is necessary to use degenerate primers to include all the possible nucleotide sequences which code the same amino acid sequence. It is important to minimize primer degeneracy since a high primer concentration is an essential requirement of a successful PCR, and a highly degenerative primer effectively reduces the concentration of individual primer sequence. In addition, a highly degenerative primer increases nonspecific amplification. We have succeeded in isolating sequences of interest with primers that contain less than 512 possible combinations. Other laboratories may have had a different experience for the maximal amount of degeneracy allowed. For example, a primer degeneracy up to 13,824 has been used, but additional screening steps to eliminate nonspecific products were needed. 6 The length of the region to be amplified should be within the limitation of the PCR, typically less than 3 kilobases (kb), although best results are obtained when the amplified product is shorter than 1 kb. Knowledge of the expected length of the PCR product(s) is helpful in the identification of the sequence of interest since PCR products may contain more than one band. Perfecting 3' Base Pairing. Because polymerase extension starts at the 3' end of the primer, it is extremely important to obtain perfect base pairing in this region in order to ensure proper priming. We find that the last 10 bases of the primers at the 3' end must have a perfect base matching. Thus, we typically include all possible combinations of the codons in this region of the primer. More mismatches are tolerated as one moves away from the last 10 bases. It may also be helpful to use codon usage to decrease degeneracy away from this region, t3 One may add linkers to the 5' end of the primers to facilitate subsequent subcloning of amplified products. However, this is not essential, and it is not desirable if the degeneracy of the primers is high. End filling by Klenow and blunt-end ligation of amplified fragments works well for subcloning. These considerations apply to the use of both cDNA and genomic DNA. However, owing to the possibility of the existence of intervening introns, the amplification from genomic DNA may not work if the length of the intron is longer than that permissible for PCR amplification. Contamination Because the PCR is so powerful in amplifying DNA sequences, a contamination of a few molecules may be amplified and become a significant fraction of the products. This is a particularly serious problem in a laboratory that handles many different types of DNAs, especially cloned
616
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
DNAs. The few molecules of the cDNAs that may exist on the bench, the hands of experimenters, and particularly the tips of pipettes can be catastrophic. Therefore, one takes precautions similar to those necessary for RNase-free conditions.7,s For example, we keep a separate set of tips, tubes, and pipettors that are used exclusively for PCR experiments. Several companies sell pipette tips designed especially for the PCR. We use long (sequencing type) pipette tips. Control Experiments Given all the precautions one may take to avoid contamination, a series of control reactions should always be performed in each experiment to evaluate the possibility of contamination. We routinely perform one reaction with both primers in the absence of DNA template in order to evaluate possible contamination of water, reagents, or pipette tips. In the case of amplification from single-stranded cDNA, one may have contamination from genomic DNA present in the original mRNA samples. In addition to treating the mRNA with RNase-free DNase, special controls are necessary to eliminate possible contamination of this type. We routinely carry out a control PCR with a sample containing all the reagents, including the mRNA, but in which cDNA was not synthesized. One may also destroy the mRNA RNase A. Enzymes and reagents of the highest quality are required to avoid amplification of contaminating DNAs. Furthermore, when exploring for amplified products in analytical DNA gels, we apply 5-20 times more reaction sample in the case of the control experiments. Experimental Protocols
General Considerations The GeneAmp PCR kit (Perkin Elmers Cetus, Norwalk, CT) which utilizes Taq polymerase is used for all of our experiments. Other thermostable polymerases are also available. The reagents and the polymerase are aliquoted immediately after receipt of the kit to minimize contamination. Separate incubation tubes and pipettors are set aside for the same reason. The PCR reaction is carded out in a DNA thermal cycler (Perkin Elmers Cetus). The experiments are carried out in the step cycle mode of the DNA thermal cycler. Typically 30-35 cycles are used; additional cycles increase only primer-dimer and background. The most sensitive parameter that may affect the specificity of amplification is the annealing temperature (T~). Higher T , preferentially allows for perfectly matched base pairing and leads to more specific amplification.
[42]
P C R CLONING OF ION CHANNEL GENE FAMILIES
617
From this point on, as we discuss the stringency of the PCR, 7"., will be the parameter under consideration. However, if one is interested in a family of related genes, lowering T,. may allow genes that have less perfect matching with primers to be amplified. In addition, when a degenerate primer is used, individual primers may have different melting temperatures owing to different nucleotide compositions. For example, we had an experience where certain genes showing a perfect base match with primers were only amplified under lower stringency conditions (see below). As expected, lower stringency leads to more nonspecific amplifications. In general, it is necessary to go through a few trials in order to identify the lowest stringency conditions where a well-defined band(s) of an expected size is obtained. Another variable of importance is the concentration of magnesium ions in the reaction. Magnesium ions affect enzymatic activity as well as the annealing of primers, the strand dissociation temperature, and the formation of primer rimers, among others, s As the importance of these factors depends on the specific reaction, it is recommended to test the PCR with any new primers or DNA at various Mg 2+ concentrations in the range of 0.5 to 5 mM. After the amplification, 5 - 10% of the product is used to run an analytical gel to determine if bands of the appropriate size appear. PCR products can be sequenced directlyS; however, if several different products of similar length are expected (as in the case discussed here), it is necessary to clone the amplified products to isolate individual sequences for analysis. We have used one of two options. One may use the entire PCR product to run a preparative gel and purify the bands of interest for cloning, or one may reamplify the initial reaction products. (If one is interested in genes expressed in low abundance, reamplification may not be a good idea. This is because the procedure may amplify abundant genes or nonspecitic products preferentially, and the relative quantities of the rare genes will be further reduced.) Two approaches were used to amplify further the product of interest. One was to dilute the PCR product, 1/1000, and repeat the same amplification reaction. Alternatively, one can gel purify the band(s) of interest and reamplify the purified fragments.
Protocols eDNA Synthesis. The following conditions are for the synthesis of first-strand cDNA starting with 1/zg of mRNA. All the reagents should be RNase free (see [17] in this volume). Mix, on ice, 1 /zg of poly(A) + m R N A with 0.5/zg of oligo(dT),2_18 (Pharmacia, Piscataway, NJ) and adjust the volume to 12/A with water that has been treated with DEPC (diethyl pyrocarbonate). Incubate the mixture at 70 ° for 10 min, chill on ice, and add the following: (1) 4#1 of 5×
618
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
reaction buffer [5× is 250 m M Tris-HC1 (pH 8.3), 375 m M KC1, 15 m M MgCI2]; (2) 2/zl of 0.1 M dithiothreitol (DTT) (Boehringer-Mannheim, Indianapolis, IN); (3) 1/zl of deoxynucleoside triphosphate (dNTP) mix [1/tl of each I00 m M d N T P (Pharmacia) with 6/zl of water]. Incubate for 2 min at 37*. Then add 1/zl (200 units) of RNase H reverse transeriptase [Bethesda Research Laboratories (BRL), Gaithersburg, MD; Cat. No. 8053SA]. Quickly remove 5/zl and mix with 1/zl of [a-32p]dCTP in a separate tube. Incubate both tubes for 1 hr at 37 °, boil for 2 min, and freeze until needed. The reaction mix can be used as PCR templates directly without precipitation of the eDNA or removing the mRNA. To estimate the yield, take 1/zl from the tube that has the radioactive dCTP and precipitate with trichloroacetie acid (TCA). Compare the TCAprecipitated counts with the total counts in 1 #1. A yield of eDNA equivalent to 5 to 15% of the starting mRNA should provide a good PCR template. To evaluate the size of the products, use 2/zl to run an alkaline agarose gel and check by autoradiography. ~4 Polymerase Chain Reaction. All the reagents are from the GeneAmp DNA amplification reagent kit. As explained earlier the kit is aliquoted on arrival. The initial aliquots are done by mixing 5 volumes of 10× reaction buffer with 1 volume of each dNTP (10 raM). Eighteen microliters of this mixture is added to 0.5-ml microcentrifuge tubes (Robin Scientific). Each tube is good for one reaction in a final volume of 100/zl. The aliquots can be stored at - 2 0 ° for up to a month. To the prealiquoted microfuge tubes add the following: (1) primers (the PCR requires high primer concentrations, which should be adjusted empiricaUy, and excess primer can lead to nonspecific amplification; we usually get good results with primer concentrations in the range of 100 ng for a nondegenerate 20-mer, or 1 - 1.5/tg for degenerate 20-mers) and (2) 1- 3 ng of eDNA, estimated from the radioactivity incorporation mentioned earlier [use 100 ng for genomic DNA and use DNA equivalent to l07 plaque-forming units (pfu) if library eDNA is used]. Adjust the volume to 99.5 /A with water. Then add Taq polymerase, 2.5 units in 0.5 /~l. (Alternatively, one can dilute the enzyme slightly, 2- to 4-fold, for the accuracy of volume measurement.) Add mineral oil, 50/zl or 3 to 4 drops, on top to prevent evaporation of the aqueous phase during the reaction. The order of adding the reagents is to minimize possible contamination. 13K. Wada, Y. Wada, H. Doi, F. Ishibashi,T. Gojobori, and T. Ikemura,NucleicAcidsRes. 19, 1981 (1991). 14j. Sambrook, E. F. Fritsch, and T. Maniatis, eds., "MolecularCloning: A Laboratory Manual," 2nd Ed. Cold SpringHarbor Laboratory,Cold SpringHarbor, New York, 1989.
[42]
F C R CLONING OF ION CHANNEL GENE FAMILIES
619
The thermocycling profile depends on the expected length of the fragment of interest and the stringency one would like to use. It is almost always necessary to go through a few trials in order to find optimal conditions for each experiment. A typical high-stringency reaction, where a fragment of 230- 500 base pairs (bp) is expected, has the following profile: 94 °, 1 min; 55 °, 1 min; 72 °, 1 rain. Typically 30-35 cycles is used. Sometimes, a 3-min denaturing period at 94 ° is applied before the cycles start to ensure complete melting, and an extension period of 7 min at 72 ° is used after the cycles are terminated to complete the extension of any incomplete products amplified during the reaction. To facilitate the removal of the oil we freeze the aqueous phase at - 2 0 ° . The reaction products are transferred to a clean tube. Take 10 #1 of the reaction product to run an analytic gel. PCR products are purified, if desired (see above). For cloning, the purified PCR products are kinased with T4 kinase. (If the kinase is shared in the laboratory, it is better to kinase the PCR fragment rather than the primers to minimize contamination.) If blunt-end ligation is used the kinased products are filled with Klenow before ligation to the desired vector. If the primers are designed with cloning sites, the products are digested with the appropriate restriction enzyme prior to ligation. Results and Discussion The following example shows the number of fragments of interest obtained from a particular experiment. By lowering the stringency of the reactions, the number of related fragments increased dramatically. In a high-stringency amplification (i.e., 94 ° for 1 min, 55 ° for 1 rain, 72 ° for 1 min), two different potassium channel sequences were identified in eDNA from PC 12 cells. With the same set of primers and eDNA, a low-stringency reaction (i.e., 94 ° for 1 min, 45 ° for 1 min, 55 ° for 1 min) produced seven different sequences. It is obvious from this example that low-stringency conditions are useful for more extensive detection of expressed genes. Whether the characterization of the expression profile is exhaustive depends on the design of primers and how much work one is willing to put into the sequencing of the subclones. Each fragment thus identified was subsequently used to isolate clones from eDNA libraries utilizing high-stringency hybridization conditions. Close to 100% of the isolated clones corresponded to the expected ones. In this regard, PCR-generated probes reduce, to a great extent, the amount of work one devotes to the characterization of false positives during lowstringency screening of libraries.
PCR
CLONING OF ION CHANNEL GENE FAMILIES
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[42] C l o n i n g o f I o n C h a n n e l G e n e F a m i l i e s U s i n g t h e Polymerase Chain Reaction B y ELEAZARC. VE6A-SAENZ DE MIERA and JEN-WEI LIN
Introduction The polymerase chain reaction (PCR), developed by Mullis et al., ~ is a method to amplify a fragment of DNA sequence by using specific primers that flank a region of interest. Thermostable polymerases are used for repeated DNA synthesis from a given template. This method has been used in various stages of ion channel cloning.2-6 When properly used, it is an extremely powerful technique, and one may obtain more than 106-fold amplification. Many reviews on the theory and applications of PCR methods are availableT,8; interested readers may obtain basic information from these sources. We describe here the use of the PCR to identify putative members of an ion channel gene family. In comparison to low-stringency hybridization, we have found the PCR to be a faster and more efficient method to discover new members of the family. For this purpose, the template for the PCR can be either genomic DNA 9 or cDNA. The cDNA may be derived from a cDNA library ~° or, as in the example described here, may be single-stranded cDNA synthesized from the mRNA of a tissue of interest. In addition, if cDNA is used as template, the amplified
K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich, Cold Spring Harbor Syrup. Quant. Biol. 51, 263 (1986). 2 G. I. Fishman, D. C. Spray, and L. A. Leinwand, J. CellBiol. 111, 589 (1990). 3 K. Folander, J. S. Smith, J. Antanavage, C. Bennett, R. B. Stein, and R. Swanson, Proc. Natl. Acad. Sci. U.S.A. 87, 2975 (1990). 4 K. Otsu, H. F. Willard, V. K. Khanna, F. Zorzato, N. M. Green, and D. H. MacLennan, J. Biol. Chem. 265, 13472 (1990). 5 j. C. L. Tseng-Crank, G.-Y. Tseng, A. Schwartz, and M. A. Tanouye, FEBS Left. 268, 63 (1990). 6 Z-Y. Zhao and R. H. Joho, Biochem. Biophys. Res. Commun. 167, 174 (1990). 7 H. A. Erlich, ed., "PCR Technology: Principles and Applications for DNA Amplification." Stockton, New York, 1989. s M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White eds., "PCR Protocols: A Guide to Methods and Applications." Academic Press, San Diego, California, 1990. 9 A. Kamb, M. Weir, B. Rudy, H. Varmus, and C. Kenyon, Proc. Natl. Acad. Sci. U.S.A. 86, 4372 (1989). 1oB. F. O'Dowd, T. Nguyen, A. Tirpak, K. R. Jarvie, Y. Israel, P. Seeman, and H. B. Niznik, FEBS Lett. 262, 8 (1990).
METHODS IN ENZYMOLOGY, VOL. 207
Copyright © 1992 by Academic Press, Inc. All fights of reproduction in any form reserved.
614
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
PCR fragments provide a profile of gene expression in a specific tixsue.H In principle, this can be done with RNA from a very small amount of tissue. ~2 In this chapter, we discuss the choice and design of PCR primers. Detailed PCR protocols provided here have worked for us and are generally applicable in most circumstances. Design of Primers The choice and design of primers constitute perhaps the most important variable for a successful PCR. To optimize the identification of distinct members of a gene family, one should amplify a region of a gene that is variable within the family. This variable region should be flanked by conserved areas where the PCR primers will be located. Therefore, the design of primers depends on previous knowledge of the sequence of some member(s) of the family. For general considerations of primer design, good sources of references are available. 7.S Some special considerations for the amplification of members of a gene family are listed below. Selecting Regionsfor Amplification. The first step is to determine the regions of the channel to be amplified. One should start with collecting as many examples of existing amino acid sequences of members of a gene family as possible so that conserved regions can be identified by sequence comparison. In case that only one or a few members of the family are known, and doubts exist about the likelihood of conservation of certain regions, knowledge or assumptions of amino acid sequence domains with specific functional roles can be used as a guide. For example, based on the speculations that the H5 region of Shaker potassium channels is involved in the selectivity for potassium ions, 9 one may design a primer from this region. (A hybrid arrest experiment may be used to evaluate the validity of the assumptions; see [41] in this volume.) In a typical PCR two conserved regions are necessary so that the sequence flanked by two primers is amplified. However, it is not necessary that both primers hybridize to sequences in the channel eDNA. For example, to amplify from library eDNA, '° one may use a primer that hybridizes to the sequence of interest and a second primer that hybridizes to sequences on a host cloning vector. Minimizing Codon Degeneracy. After conserved areas are identified, the amino acid sequence of the regions should be analyzed to locate a stretch of amino acid sequence with minimal codon degeneracy. (PCR primers typically have a length of 18 to 30 nucleotides. 8) Given the asit E. Vega-Saenz de Micra, N. Chiu, K. Sen, D. Lau, J. W. Lin, and B. Rudy, Biophys. J. 59, 197a (1991). 12 H. H. Li, U. B. Gyllensten, X. F. Cui, R. K. Saiki, and H. A. Erfieh, Nature (London) 335, 414(1988).
[42]
P C R CLONING OF ION CHANNEL GENE FAMILIES
615
sumption that only amino acid sequence is conserved in the course of evolution, it is necessary to use degenerate primers to include all the possible nucleotide sequences which code the same amino acid sequence. It is important to minimize primer degeneracy since a high primer concentration is an essential requirement of a successful PCR, and a highly degenerative primer effectively reduces the concentration of individual primer sequence. In addition, a highly degenerative primer increases nonspecific amplification. We have succeeded in isolating sequences of interest with primers that contain less than 512 possible combinations. Other laboratories may have had a different experience for the maximal amount of degeneracy allowed. For example, a primer degeneracy up to 13,824 has been used, but additional screening steps to eliminate nonspecific products were needed. 6 The length of the region to be amplified should be within the limitation of the PCR, typically less than 3 kilobases (kb), although best results are obtained when the amplified product is shorter than 1 kb. Knowledge of the expected length of the PCR product(s) is helpful in the identification of the sequence of interest since PCR products may contain more than one band. Perfecting 3' Base Pairing. Because polymerase extension starts at the 3' end of the primer, it is extremely important to obtain perfect base pairing in this region in order to ensure proper priming. We find that the last 10 bases of the primers at the 3' end must have a perfect base matching. Thus, we typically include all possible combinations of the codons in this region of the primer. More mismatches are tolerated as one moves away from the last 10 bases. It may also be helpful to use codon usage to decrease degeneracy away from this region, t3 One may add linkers to the 5' end of the primers to facilitate subsequent subcloning of amplified products. However, this is not essential, and it is not desirable if the degeneracy of the primers is high. End filling by Klenow and blunt-end ligation of amplified fragments works well for subcloning. These considerations apply to the use of both cDNA and genomic DNA. However, owing to the possibility of the existence of intervening introns, the amplification from genomic DNA may not work if the length of the intron is longer than that permissible for PCR amplification. Contamination Because the PCR is so powerful in amplifying DNA sequences, a contamination of a few molecules may be amplified and become a significant fraction of the products. This is a particularly serious problem in a laboratory that handles many different types of DNAs, especially cloned
616
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
DNAs. The few molecules of the cDNAs that may exist on the bench, the hands of experimenters, and particularly the tips of pipettes can be catastrophic. Therefore, one takes precautions similar to those necessary for RNase-free conditions.7,s For example, we keep a separate set of tips, tubes, and pipettors that are used exclusively for PCR experiments. Several companies sell pipette tips designed especially for the PCR. We use long (sequencing type) pipette tips. Control Experiments Given all the precautions one may take to avoid contamination, a series of control reactions should always be performed in each experiment to evaluate the possibility of contamination. We routinely perform one reaction with both primers in the absence of DNA template in order to evaluate possible contamination of water, reagents, or pipette tips. In the case of amplification from single-stranded cDNA, one may have contamination from genomic DNA present in the original mRNA samples. In addition to treating the mRNA with RNase-free DNase, special controls are necessary to eliminate possible contamination of this type. We routinely carry out a control PCR with a sample containing all the reagents, including the mRNA, but in which cDNA was not synthesized. One may also destroy the mRNA RNase A. Enzymes and reagents of the highest quality are required to avoid amplification of contaminating DNAs. Furthermore, when exploring for amplified products in analytical DNA gels, we apply 5-20 times more reaction sample in the case of the control experiments. Experimental Protocols
General Considerations The GeneAmp PCR kit (Perkin Elmers Cetus, Norwalk, CT) which utilizes Taq polymerase is used for all of our experiments. Other thermostable polymerases are also available. The reagents and the polymerase are aliquoted immediately after receipt of the kit to minimize contamination. Separate incubation tubes and pipettors are set aside for the same reason. The PCR reaction is carded out in a DNA thermal cycler (Perkin Elmers Cetus). The experiments are carried out in the step cycle mode of the DNA thermal cycler. Typically 30-35 cycles are used; additional cycles increase only primer-dimer and background. The most sensitive parameter that may affect the specificity of amplification is the annealing temperature (T~). Higher T , preferentially allows for perfectly matched base pairing and leads to more specific amplification.
[42]
P C R CLONING OF ION CHANNEL GENE FAMILIES
617
From this point on, as we discuss the stringency of the PCR, 7"., will be the parameter under consideration. However, if one is interested in a family of related genes, lowering T,. may allow genes that have less perfect matching with primers to be amplified. In addition, when a degenerate primer is used, individual primers may have different melting temperatures owing to different nucleotide compositions. For example, we had an experience where certain genes showing a perfect base match with primers were only amplified under lower stringency conditions (see below). As expected, lower stringency leads to more nonspecific amplifications. In general, it is necessary to go through a few trials in order to identify the lowest stringency conditions where a well-defined band(s) of an expected size is obtained. Another variable of importance is the concentration of magnesium ions in the reaction. Magnesium ions affect enzymatic activity as well as the annealing of primers, the strand dissociation temperature, and the formation of primer rimers, among others, s As the importance of these factors depends on the specific reaction, it is recommended to test the PCR with any new primers or DNA at various Mg 2+ concentrations in the range of 0.5 to 5 mM. After the amplification, 5 - 10% of the product is used to run an analytical gel to determine if bands of the appropriate size appear. PCR products can be sequenced directlyS; however, if several different products of similar length are expected (as in the case discussed here), it is necessary to clone the amplified products to isolate individual sequences for analysis. We have used one of two options. One may use the entire PCR product to run a preparative gel and purify the bands of interest for cloning, or one may reamplify the initial reaction products. (If one is interested in genes expressed in low abundance, reamplification may not be a good idea. This is because the procedure may amplify abundant genes or nonspecitic products preferentially, and the relative quantities of the rare genes will be further reduced.) Two approaches were used to amplify further the product of interest. One was to dilute the PCR product, 1/1000, and repeat the same amplification reaction. Alternatively, one can gel purify the band(s) of interest and reamplify the purified fragments.
Protocols eDNA Synthesis. The following conditions are for the synthesis of first-strand cDNA starting with 1/zg of mRNA. All the reagents should be RNase free (see [17] in this volume). Mix, on ice, 1 /zg of poly(A) + m R N A with 0.5/zg of oligo(dT),2_18 (Pharmacia, Piscataway, NJ) and adjust the volume to 12/A with water that has been treated with DEPC (diethyl pyrocarbonate). Incubate the mixture at 70 ° for 10 min, chill on ice, and add the following: (1) 4#1 of 5×
618
PURIFICATION OF ION CHANNEL PROTEINS AND GENES
[42]
reaction buffer [5× is 250 m M Tris-HC1 (pH 8.3), 375 m M KC1, 15 m M MgCI2]; (2) 2/zl of 0.1 M dithiothreitol (DTT) (Boehringer-Mannheim, Indianapolis, IN); (3) 1/zl of deoxynucleoside triphosphate (dNTP) mix [1/tl of each I00 m M d N T P (Pharmacia) with 6/zl of water]. Incubate for 2 min at 37*. Then add 1/zl (200 units) of RNase H reverse transeriptase [Bethesda Research Laboratories (BRL), Gaithersburg, MD; Cat. No. 8053SA]. Quickly remove 5/zl and mix with 1/zl of [a-32p]dCTP in a separate tube. Incubate both tubes for 1 hr at 37 °, boil for 2 min, and freeze until needed. The reaction mix can be used as PCR templates directly without precipitation of the eDNA or removing the mRNA. To estimate the yield, take 1/zl from the tube that has the radioactive dCTP and precipitate with trichloroacetie acid (TCA). Compare the TCAprecipitated counts with the total counts in 1 #1. A yield of eDNA equivalent to 5 to 15% of the starting mRNA should provide a good PCR template. To evaluate the size of the products, use 2/zl to run an alkaline agarose gel and check by autoradiography. ~4 Polymerase Chain Reaction. All the reagents are from the GeneAmp DNA amplification reagent kit. As explained earlier the kit is aliquoted on arrival. The initial aliquots are done by mixing 5 volumes of 10× reaction buffer with 1 volume of each dNTP (10 raM). Eighteen microliters of this mixture is added to 0.5-ml microcentrifuge tubes (Robin Scientific). Each tube is good for one reaction in a final volume of 100/zl. The aliquots can be stored at - 2 0 ° for up to a month. To the prealiquoted microfuge tubes add the following: (1) primers (the PCR requires high primer concentrations, which should be adjusted empiricaUy, and excess primer can lead to nonspecific amplification; we usually get good results with primer concentrations in the range of 100 ng for a nondegenerate 20-mer, or 1 - 1.5/tg for degenerate 20-mers) and (2) 1- 3 ng of eDNA, estimated from the radioactivity incorporation mentioned earlier [use 100 ng for genomic DNA and use DNA equivalent to l07 plaque-forming units (pfu) if library eDNA is used]. Adjust the volume to 99.5 /A with water. Then add Taq polymerase, 2.5 units in 0.5 /~l. (Alternatively, one can dilute the enzyme slightly, 2- to 4-fold, for the accuracy of volume measurement.) Add mineral oil, 50/zl or 3 to 4 drops, on top to prevent evaporation of the aqueous phase during the reaction. The order of adding the reagents is to minimize possible contamination. 13K. Wada, Y. Wada, H. Doi, F. Ishibashi,T. Gojobori, and T. Ikemura,NucleicAcidsRes. 19, 1981 (1991). 14j. Sambrook, E. F. Fritsch, and T. Maniatis, eds., "MolecularCloning: A Laboratory Manual," 2nd Ed. Cold SpringHarbor Laboratory,Cold SpringHarbor, New York, 1989.
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F C R CLONING OF ION CHANNEL GENE FAMILIES
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The thermocycling profile depends on the expected length of the fragment of interest and the stringency one would like to use. It is almost always necessary to go through a few trials in order to find optimal conditions for each experiment. A typical high-stringency reaction, where a fragment of 230- 500 base pairs (bp) is expected, has the following profile: 94 °, 1 min; 55 °, 1 min; 72 °, 1 rain. Typically 30-35 cycles is used. Sometimes, a 3-min denaturing period at 94 ° is applied before the cycles start to ensure complete melting, and an extension period of 7 min at 72 ° is used after the cycles are terminated to complete the extension of any incomplete products amplified during the reaction. To facilitate the removal of the oil we freeze the aqueous phase at - 2 0 ° . The reaction products are transferred to a clean tube. Take 10 #1 of the reaction product to run an analytic gel. PCR products are purified, if desired (see above). For cloning, the purified PCR products are kinased with T4 kinase. (If the kinase is shared in the laboratory, it is better to kinase the PCR fragment rather than the primers to minimize contamination.) If blunt-end ligation is used the kinased products are filled with Klenow before ligation to the desired vector. If the primers are designed with cloning sites, the products are digested with the appropriate restriction enzyme prior to ligation. Results and Discussion The following example shows the number of fragments of interest obtained from a particular experiment. By lowering the stringency of the reactions, the number of related fragments increased dramatically. In a high-stringency amplification (i.e., 94 ° for 1 min, 55 ° for 1 rain, 72 ° for 1 min), two different potassium channel sequences were identified in eDNA from PC 12 cells. With the same set of primers and eDNA, a low-stringency reaction (i.e., 94 ° for 1 min, 45 ° for 1 min, 55 ° for 1 min) produced seven different sequences. It is obvious from this example that low-stringency conditions are useful for more extensive detection of expressed genes. Whether the characterization of the expression profile is exhaustive depends on the design of primers and how much work one is willing to put into the sequencing of the subclones. Each fragment thus identified was subsequently used to isolate clones from eDNA libraries utilizing high-stringency hybridization conditions. Close to 100% of the isolated clones corresponded to the expected ones. In this regard, PCR-generated probes reduce, to a great extent, the amount of work one devotes to the characterization of false positives during lowstringency screening of libraries.
