- Email: [email protected]
[31] Reverse transcription of mRNA by Thermus aquaticus DNA polymerase followed by polymerase chain reaction amplification
[31]
REVERSETRANSCRIPTIONOF mRNA By Taq DNA POLYMERASE
413
hybrids 70M11 and 80H7. We also found one example, 81P8, of the opposite type of discordance, for which we fail to detect a chromosome that is scored positive in the cell line. These types of discordancies may arise when the target sequence, like BEK, is located at the very end of a chromosome, where small truncations are less noticeable. It is likely that when the more sensitive PCR techniques are used in characterization of the panels, both type of discordancies will diminish in number. It is always useful, if possible, to verify the results of PCR-generated assignment by comparison to the results obtained for another gene mapped to the same chromosome. Theoretically, two genes on the same chromosome should cosegregate. Similarly, the product should be available from the appropriate chromosome specific genomic DNA library. Even with the extra controls and verifications, chromosome assignment by PCR technique is much faster than by conventional methods and is much more conservative of the valuable somatic cell hybrid DNA. Acknowledgments The authors thank Dr. Stephen O'Brien and Dr. WilliamModifor the gift of somatic cell hybrid DNAs. We thank Robin McCormickand PatriciaGallagherfor excellentpreparation of the manuscript.
[31] R e v e r s e T r a n s c r i p t i o n o f m R N A b y Thermus aquaticus DNA Polymerase followed by Polymerase Chain Reaction Amplification
By MICHAEL D. JONES Introduction The structural analysis of messenger RNA (mRNA) plays an important role in the examination of gene structure and expression. The key step is the reverse transcription of mRNA into complementary DNA (cDNA), which subsequently can be cloned and analyzed. Central to this process are the reverse transcriptases (RNA-dependent DNA polymerases, EC 2.7.7.49). One difficulty that can prevent the efficient copying of RNA sequences into cDNA is the ability of single-stranded RNA to adopt stable intramolecular stem-loop structures. This can cause pausing of the reverse transcriptase and thus result in premature termination of the reverse transcript.
METHODS IN ENZYMOLOGY, VOL. 218
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PCR FOR AMPLIFYINGAND MANIPULATINGDNA
[31]
Such secondary structure may account for the relatively poor yields of fulllength c D N A clones obtained in the construction of recombinant c D N A libraries. One solution to this problem would be to perform the reverse transcription reaction at an elevated temperature at which such structures would be disrupted, allowing the e n z y m e to copy through to the 5' end of the R N A molecule. L o e b et al.l reported that Escherichia coli D N A polymerase has the potential to use R N A as a template for the synthesis of D N A in vitro, and Chien et al. 2 have alluded to the possibility that Thermus aquaticus D N A polymerase may possess reverse transcriptase activity. The advent of the polymerase chain reaction (PCR) and the subsequent commercial availability of thermostable D N A polymerases 3 permitted an investigation into the utilization of T. aquaticus D N A polymerase to copy R N A into cDNA. 4
Materials Enzymes Thermus aquaticus D N A polymerase and AmpliTaq (cloned T. aquaticus D N A polymerase) are from Perkin-Elmer Cetus (Norwalk, CT), avian
myeloblastosis virus (AMV) reverse transcriptase is from Anglian Limited (Essex, UK) and IBI, Limited (New Haven, CT) RNase-free DNase is purchased from Stratagene (La Jolla, CA), RNasin is from Promega (Madison, WI), and RNase A is from Sigma (St. Louis, MO). Reagents
d N T P s are from P h a r m a c i a - L K B (Piscataway, N J) and are dissolved in 10 m M Tris-HC1 (pH 8.0), 0.1 m M ethylenediaminetetraacetic acid (EDTA), to 10 m M and stored at - 2 0 °. The oligodeoxynucleotide primers are synthesized on an Applied Biosystems (Foster City, CA) 380B D N A synthesizer, deprotected, precipitated with ethanol, and finally dissolved in water at - I - 2 / z g / / x l . T h e y are used without further purification. The sequences of the primers are as follow: primer G, 5 ' - G G G A A G G A G G G T G G C C G T G - 3 ' ; primer F, 5'-CTTCAACCCCGAGGAGT-3'4,5; primer 6SP 1, 5 ' - C G C G T T C G T T T A A C A T A T G G - 3 ' ; and primer 6SP2, 5 '-GAAT1L. A. Loeb, K. D. Tartof, and E. C. Travaglini, Nature (London) NewBiol. 242, 66 (1973). 2 A. Chien, D. B. Edgar, and J. M. Trela, J. Bacteriol. 127, 1550 (1976). 3 R. K. Saiki, D. H. Gelfand, S. Stoffel, S. Scharf, R. Huguchi, G. T. Horn, K. B. Mullis, and H. A. Erlich, Science 239, 487 (1988). 4 M. D. Jones and N. S. Foulkes, Nucleic Acids Res. 17, 8387 (1989). M. G. Persico, G. Viglietto, G. Martini, D. Toniolo, G. Paonessa, C. Moscatelli, R. Dono, T. Vulliamy, L. Luzzatto, and M. D'Urso, Nucleic Acids Res. 14, 2511, 7822 (1986).
[31]
REVERSETRANSCRIPTION OF m R N A BY
Taq D N A
POLYMERASE
415
CAGCATCGGTTACGTG-3'. SeaKem ME agarose is purchased from FMC Corporation (Rockland, ME). Size markers for agarose gel electrophoresis are prepared from either pBS or pBluescript K S ( - ) , purchased from Stratagene, by digestion with Taq I and SauAI, separately, and then mixed together in equimolar amounts. 4
Solutions RNA lysis buffer: 140 mM NaC1, 1.5 mM MgC12 , 10 mM Tris-HCl (pH 8.0), 0.5% (v/v) Nonidet P-40 (NP-40), 0.15% (w/v) Macaloid (Steetly Minerals Ltd.) RT buffer (10 x ): 500 mM KCI, 80 mM MgC1z , 500 mM Tris-HCl (pH 8.3) 6 PCR buffer (I0 x ): 500 mM KC1, 20 mM MgC12 , 100 mM Tris-HCl (pH 8.3) TBE buffer (10 x): 108 g of Tris base, 55 g of boric acid, 9.3 g of Na2EDTA per liter of water dNTP mix: 2.5 mM concentrations of each dNTP
mRNA Total cytoplasmic RNA from HeLa cells and HHV-6 infected HSB-2 cells are prepared by the method of Favaloro et al. 7 Essentially, washed cells are resuspended in lysis buffer, and after 5 min on ice the nuclei are pelleted and the supernatant removed. The supernatant is extracted three times with phenol-chloroform. Cytoplasmic RNA is precipitated with ethanol and dissolved in 10 mM Tris-HC1 (pH 7.0) at approximately 1-5 mg/ml and stored at - 7 0 °. Methods 1. RNA, 1-5/xg, in 5/A diethyl pyrocarbonate (DEPC)-treated water is heated to 90 ° for 2-5 min and then rapidly chilled on ice. Two microliters of 10 × RT buffer or 10 × PCR buffer is added, together with 8 ~1 of 2.5 mM dNTP mix (final concentration for reverse transcription is 1 mM for each dNTP), 40 units of RNasin, 50 ng of the reverse oligodeoxynucleotide primer, and either 40 units of AMV reverse transcriptase or 2.5 units of AmpliTaq (T. aquaticus DNA polymerase). Then DEPC-treated water is added to a final volume of 20 /zl. For AMV reverse transcriptase, the reaction mixture is incubated at 42 ° for 60 min and for T. aquaticus DNA polymerase at 68-72 ° for 30-60 min. The reaction mixture is then heated to 90° for 5 min and rapidly chilled on ice. 6 C. J. Watson and J. F. Jackson, in "DNA Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 1, p. 79. IRL Press, Oxford, 1985.
7j. Favaloro, R. Treisman, and R. Kamen, this series, Vol. 65, p. 718.
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A M
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FIG. 1. G6PD reverse transcription by AMV reverse transcriptase and T. aquatictts DNA polymerase. Aliquots of HeLa cell total cytoplasmic RNA (-5/zg) were treated as follows: lane 3, with 20 units of AMV reverse transcriptase and 1/zg of primer G in 20 ~1 RT buffer at 42° for 45 min, and subsequent PCR amplification, for 30 cycles, with 1/zg each of primers F and G, and 2.5 units of T. aquaticus DNA polymerase; lane 4, as for lane 3 but with prior incubation with 10 ~g of RNase A; lane 5, reverse transcription with 2.5 units of T. aquaticus DNA polymerase in 20/zl of PCR buffer with 1 ~g of primer G, for 30 min at 68°. The sample was then treated to PCR amplification as for lane 3. Lane 6, direct PCR amplification with 2.5 units of T. aquaticus DNA polymerase, 1/~g each of primers G and F in 50/zl PCR buffer. Lanes 1 and 2 were plasmid cDNA and genomic DNA clones ( - 5 0 ng each), respectively, amplified directly. Lanes M are size markers of an equimolar mixture of TaqI- and Sau3AI-
[31]
REVERSETRANSCRIPTIONOF mRNA BY Taq DNA POLYMERASE
417
2. Ten microliters of the c D N A mixture from step I a b o v e is amplified by PCR. The buffer is adjusted to 50 m M KC1, 2 m M MgCI2, 10 m M TrisHCI (pH 8.3) (1 x PCR buffer), 200 tzM for each dNTP. One m i c r o g r a m of both forward and reverse oligodeoxynucleotide primers and 2.5 units of AmpliTaq are added, and the final volume adjusted to 50/zl with water. Mineral oil (50 tzl) is overlaid on top of the reaction mix to prevent evaporation during the PCR cycles. 3. The reaction mixture is subjected to amplification for 30-35 cycles. The actual p a r a m e t e r s will vary depending on the length of the target sequence and the GC content of the primers. The values used in the examples shown here were as follow: 94 °, 1 min; 55 °, 1 min; 72 °, 4 min, for 30 cycles using a Perkin-Elmer Cetus D N A thermal cycler (Fig. 1) and 94 °, 30 sec; 55 °, 30 sec; 72 °, 2 min, for 30-35 cycles using an M J Research (Watertown, MA) p r o g r a m m a b l e thermal controller (Fig. 2). The last cycle includes an incubation at 72 ° for an extra 5 min. 4. After completion of the PCR cycles, the mineral oil is carefully extracted twice with 500/zl of chloroform and the aqueous phase stored at - 20 °. Eight microliters of the reaction mix is analyzed by agarose gel electrophoresis. Fifty-milliliter agarose gels (1.4-1.6% agarose) in 1 x T B E buffer, containing 50/zg/ml ethidium bromide, are run submarine fashion at 60-mA constant current. The PCR-amplified D N A fragments can be analyzed by standard Southern blot analysis 8 using cloned plasmid sequences containing the PCR target sequences. Discussion The experiments shown were designed to reverse transcribe m R N A into c D N A with primers specific for the human glucose-6-phosphate dehydrogenase gene (G6PD) 4'5 and a conserved viral gene from human herpesvirus 6 (HHV-6). 9 The primers were chosen in each case to cross an intron such that amplification from m R N A could be distinguished from genomic D N A . R e v e r s e transcription of G6PD-specific R N A with either A M V 8 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Press, Cold Spring Harbor, New York, 1982. 9 G. L. Lawrence, M. Chee, M. A. Craxton, U. A. Gompels, R. W. Honess, and B. G. Barrell, J. Virol. 64, 287 (1990).
digested pBS. (A) Ethidium bromide-stained agarose gel; (B) and (C) are two different exposures of the Southern blot probed with the plasmid cDNA clone. Primers G and F amplify a genomic DNA fragment of 657 bp and an mRNA fragment of 358 bp. (Reproduced from Jones and Foulkes4 with permission of Oxford University Press.)
418
P C R FOR AMPLIFYING AND MANIPULATING D N A M
1
2
3
4
5
6
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M
Genomic 1444-1030 740512 465-341 258 --
.,--cDNA
189 -105
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FIG. 2. HHV-6 reverse transcription by AMV reverse transcriptase and T. aquaticus DNA polymerase. Aliquots of total cytoplasmic RNA from HHV-6-infected HSB-2 cells, -2/~g, were reverse transcribed in 1 x PCR buffer (final volume, 20 t~l) with 50 ng primer 6SPI, 40 units AMV reverse transcriptase at 42° for 60 min (lanes 1-3) or 2.5 units of AmpliTaq at 72° for 60 min (lanes 4-6). Samples (10/~1) were then submitted to amplification in 1 × PCR buffer (50-~1 final volume) with 0.5/~g each of primers 6SPI and 6SP2, and 2.5 units of AmpliTaq. Samples were amplified through 30 cycles (lanes 1-3) or 35 cycles (lanes 4-6). In lanes 2 and 5 the RNA was incubated with 10 ~g of RNase A for 60 min at 37° prior to reverse transcription, and lanes 3 and 6 were pretreated with 10 units of RNase-free DNase for 60 rain at 37°. Lanes M are size markers of an equimolar mixture of TaqI- and Sau3AIdigested pBluescript KS( - ). The arrowed genomic PCR fragment is 3623 bp in size and the cDNA fragment is 426 bp.
r e v e r s e t r a n s c r i p t a s e o r T. aquaticus D N A p o l y m e r a s e r e s u l t e d a f t e r P C R a m p l i f i c a t i o n in a s m e a r o f D N A f r a g m e n t s (Fig. 1A). S p e c i f i c a m p l i f i c a t i o n o f t h e G 6 P D c D N A s e q u e n c e s w a s c o n f i r m e d b y S o u t h e r n b l o t t i n g (Fig. 1B a n d C). N o c o n t a m i n a t i n g n u c l e a r g e n o m i c D N A w a s d e t e c t e d , as s h o w n b y t h e a b s e n c e o f a D N A b a n d o f 657 bp. R N a s e d i g e s t i o n p r i o r to P C R c o m p l e t e l y d e s t r o y e d a n y G 6 P D c D N A a m p l i f i c a t i o n (Fig. 1, l a n e 4). R e v e r s e t r a n s c r i p t i o n b y T. aquaticus D N A p o l y m e r a s e is c a p a b l e o f g i v i n g r i s e to l a r g e f r a g m e n t s , as s o m e o f t h e a r t i f a c t u a l b a n d s g e n e r a t e d a r e g r e a t e r t h a n 1000 b p in size (Fig. 1A, l a n e 5). H u m a n h e r p e s v i r u s 6 i n f e c t i o n o f cells l e a d s to v i r a l p a r t i c l e s in t h e cytoplasm, and preparation of cytoplasmic RNA concomitantly isolates v i r a l g e n o m i c D N A . T h e i n t r o n p r e s e n t in t h e g e n e is o v e r 3000 b p in size.9 T h i s " c o n t a m i n a t i n g " D N A w a s c o n c l u s i v e l y s e e n in t h e R N A s a m p l e and was successfully eliminated by pretreatment of the RNA with DNase
[31]
REVERSETRANSCRIPTIONOF mRNA BY Taq DNA POLYMERASE
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(Fig. 2). This in effect released primers sequestered by genomic DNA, which could be utilized to amplify c D N A (Fig. 2, compare lanes 1 and 3, and 4 and 6). The correct c D N A band was confirmed by Southern blot analysis with an H H V - 6 recombinant plasmid clone (data not shown). Thus, if contaminating D N A is present in an RNA sample, DNase digestion will improve the yield of PCR-amplified cDNA. The efficiency of reverse transcription by T. aquaticus DNA polymerase is low compared with AMV reverse transcriptase, approximately - 1 % . This is not surprising, as T. aquaticus DNA polymerase has evolved to utilize a D N A template rather than a RNA one. A simple way to o v e r c o m e the inefficient reverse transcription step is to perform the PCR amplification for 40 or more cycles. The gene for T. aquaticus DNA polymerase has been cloned and expressed in E. coli, I° and with sitedirected mutagenesis it should be possible to engineer an enzyme with an improved reverse transcriptase activity. Analysis of the specificities of other thermostable D N A polymerases, for example, the enzyme isolated from Thermus thermophilus, may reveal polymerases with better reverse transcriptase activity.ll A requirement for 2-3 mM MgC12 was found for the reverse transcription of the G6PD sequences by T. aquaticus DNA polymerase. 4 Thus, as with PCR, care must be exercised in the magnesium ion concentration in the reaction buffer. Investigation into the replacement of magnesium with manganese may improve the reverse transcriptase activity of T. aquaticus D N A polymerase. 11 Tse and Forget 12 have also shown the ability of T. aquaticus D N A polymerase to carry out reverse transcriptions. They obtained reverse transcription by directly amplifying the RNA under standard PCR conditions, without a transcription preincubation step. We have not been able to amplify R N A directly by PCR (Fig. 1, lane 6), 4 but this may be a reflection of the actual R N A sequences involved. In conclusion, T. aquaticus D N A polymerase has been shown to possess reverse transcriptase activity and this e n z y m e (or engineered variants and other thermostable polymerases) should prove extremely useful for the analysis of m R N A structure. Acknowledgments The work described was supported by grants from the WellcomeTrust, Cancer Research Campaign, the Nuffield Foundation, and the Society for General Microbiology. l0 F. C. Lawyer, S. Stoffel, R. K. Saiki, K. Myambo, R. Drummond, and D. H. Gelfand, J. Biol. Chem. 264, 6427 (1989). ii Perkin-Elmer Cetus is marketing T. thermophilus DNA polymerase as a single enzyme for both reverse transcription and PCR amplification. t2 W. T. Tse and B. G. Forget, Gene 88, 293 (1990).
REVERSETRANSCRIPTIONOF mRNA By Taq DNA POLYMERASE
413
hybrids 70M11 and 80H7. We also found one example, 81P8, of the opposite type of discordance, for which we fail to detect a chromosome that is scored positive in the cell line. These types of discordancies may arise when the target sequence, like BEK, is located at the very end of a chromosome, where small truncations are less noticeable. It is likely that when the more sensitive PCR techniques are used in characterization of the panels, both type of discordancies will diminish in number. It is always useful, if possible, to verify the results of PCR-generated assignment by comparison to the results obtained for another gene mapped to the same chromosome. Theoretically, two genes on the same chromosome should cosegregate. Similarly, the product should be available from the appropriate chromosome specific genomic DNA library. Even with the extra controls and verifications, chromosome assignment by PCR technique is much faster than by conventional methods and is much more conservative of the valuable somatic cell hybrid DNA. Acknowledgments The authors thank Dr. Stephen O'Brien and Dr. WilliamModifor the gift of somatic cell hybrid DNAs. We thank Robin McCormickand PatriciaGallagherfor excellentpreparation of the manuscript.
[31] R e v e r s e T r a n s c r i p t i o n o f m R N A b y Thermus aquaticus DNA Polymerase followed by Polymerase Chain Reaction Amplification
By MICHAEL D. JONES Introduction The structural analysis of messenger RNA (mRNA) plays an important role in the examination of gene structure and expression. The key step is the reverse transcription of mRNA into complementary DNA (cDNA), which subsequently can be cloned and analyzed. Central to this process are the reverse transcriptases (RNA-dependent DNA polymerases, EC 2.7.7.49). One difficulty that can prevent the efficient copying of RNA sequences into cDNA is the ability of single-stranded RNA to adopt stable intramolecular stem-loop structures. This can cause pausing of the reverse transcriptase and thus result in premature termination of the reverse transcript.
METHODS IN ENZYMOLOGY, VOL. 218
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PCR FOR AMPLIFYINGAND MANIPULATINGDNA
[31]
Such secondary structure may account for the relatively poor yields of fulllength c D N A clones obtained in the construction of recombinant c D N A libraries. One solution to this problem would be to perform the reverse transcription reaction at an elevated temperature at which such structures would be disrupted, allowing the e n z y m e to copy through to the 5' end of the R N A molecule. L o e b et al.l reported that Escherichia coli D N A polymerase has the potential to use R N A as a template for the synthesis of D N A in vitro, and Chien et al. 2 have alluded to the possibility that Thermus aquaticus D N A polymerase may possess reverse transcriptase activity. The advent of the polymerase chain reaction (PCR) and the subsequent commercial availability of thermostable D N A polymerases 3 permitted an investigation into the utilization of T. aquaticus D N A polymerase to copy R N A into cDNA. 4
Materials Enzymes Thermus aquaticus D N A polymerase and AmpliTaq (cloned T. aquaticus D N A polymerase) are from Perkin-Elmer Cetus (Norwalk, CT), avian
myeloblastosis virus (AMV) reverse transcriptase is from Anglian Limited (Essex, UK) and IBI, Limited (New Haven, CT) RNase-free DNase is purchased from Stratagene (La Jolla, CA), RNasin is from Promega (Madison, WI), and RNase A is from Sigma (St. Louis, MO). Reagents
d N T P s are from P h a r m a c i a - L K B (Piscataway, N J) and are dissolved in 10 m M Tris-HC1 (pH 8.0), 0.1 m M ethylenediaminetetraacetic acid (EDTA), to 10 m M and stored at - 2 0 °. The oligodeoxynucleotide primers are synthesized on an Applied Biosystems (Foster City, CA) 380B D N A synthesizer, deprotected, precipitated with ethanol, and finally dissolved in water at - I - 2 / z g / / x l . T h e y are used without further purification. The sequences of the primers are as follow: primer G, 5 ' - G G G A A G G A G G G T G G C C G T G - 3 ' ; primer F, 5'-CTTCAACCCCGAGGAGT-3'4,5; primer 6SP 1, 5 ' - C G C G T T C G T T T A A C A T A T G G - 3 ' ; and primer 6SP2, 5 '-GAAT1L. A. Loeb, K. D. Tartof, and E. C. Travaglini, Nature (London) NewBiol. 242, 66 (1973). 2 A. Chien, D. B. Edgar, and J. M. Trela, J. Bacteriol. 127, 1550 (1976). 3 R. K. Saiki, D. H. Gelfand, S. Stoffel, S. Scharf, R. Huguchi, G. T. Horn, K. B. Mullis, and H. A. Erlich, Science 239, 487 (1988). 4 M. D. Jones and N. S. Foulkes, Nucleic Acids Res. 17, 8387 (1989). M. G. Persico, G. Viglietto, G. Martini, D. Toniolo, G. Paonessa, C. Moscatelli, R. Dono, T. Vulliamy, L. Luzzatto, and M. D'Urso, Nucleic Acids Res. 14, 2511, 7822 (1986).
[31]
REVERSETRANSCRIPTION OF m R N A BY
Taq D N A
POLYMERASE
415
CAGCATCGGTTACGTG-3'. SeaKem ME agarose is purchased from FMC Corporation (Rockland, ME). Size markers for agarose gel electrophoresis are prepared from either pBS or pBluescript K S ( - ) , purchased from Stratagene, by digestion with Taq I and SauAI, separately, and then mixed together in equimolar amounts. 4
Solutions RNA lysis buffer: 140 mM NaC1, 1.5 mM MgC12 , 10 mM Tris-HCl (pH 8.0), 0.5% (v/v) Nonidet P-40 (NP-40), 0.15% (w/v) Macaloid (Steetly Minerals Ltd.) RT buffer (10 x ): 500 mM KCI, 80 mM MgC1z , 500 mM Tris-HCl (pH 8.3) 6 PCR buffer (I0 x ): 500 mM KC1, 20 mM MgC12 , 100 mM Tris-HCl (pH 8.3) TBE buffer (10 x): 108 g of Tris base, 55 g of boric acid, 9.3 g of Na2EDTA per liter of water dNTP mix: 2.5 mM concentrations of each dNTP
mRNA Total cytoplasmic RNA from HeLa cells and HHV-6 infected HSB-2 cells are prepared by the method of Favaloro et al. 7 Essentially, washed cells are resuspended in lysis buffer, and after 5 min on ice the nuclei are pelleted and the supernatant removed. The supernatant is extracted three times with phenol-chloroform. Cytoplasmic RNA is precipitated with ethanol and dissolved in 10 mM Tris-HC1 (pH 7.0) at approximately 1-5 mg/ml and stored at - 7 0 °. Methods 1. RNA, 1-5/xg, in 5/A diethyl pyrocarbonate (DEPC)-treated water is heated to 90 ° for 2-5 min and then rapidly chilled on ice. Two microliters of 10 × RT buffer or 10 × PCR buffer is added, together with 8 ~1 of 2.5 mM dNTP mix (final concentration for reverse transcription is 1 mM for each dNTP), 40 units of RNasin, 50 ng of the reverse oligodeoxynucleotide primer, and either 40 units of AMV reverse transcriptase or 2.5 units of AmpliTaq (T. aquaticus DNA polymerase). Then DEPC-treated water is added to a final volume of 20 /zl. For AMV reverse transcriptase, the reaction mixture is incubated at 42 ° for 60 min and for T. aquaticus DNA polymerase at 68-72 ° for 30-60 min. The reaction mixture is then heated to 90° for 5 min and rapidly chilled on ice. 6 C. J. Watson and J. F. Jackson, in "DNA Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 1, p. 79. IRL Press, Oxford, 1985.
7j. Favaloro, R. Treisman, and R. Kamen, this series, Vol. 65, p. 718.
416
P C R FOR AMPLIFYING AND MANIPULATING D N A
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FIG. 1. G6PD reverse transcription by AMV reverse transcriptase and T. aquatictts DNA polymerase. Aliquots of HeLa cell total cytoplasmic RNA (-5/zg) were treated as follows: lane 3, with 20 units of AMV reverse transcriptase and 1/zg of primer G in 20 ~1 RT buffer at 42° for 45 min, and subsequent PCR amplification, for 30 cycles, with 1/zg each of primers F and G, and 2.5 units of T. aquaticus DNA polymerase; lane 4, as for lane 3 but with prior incubation with 10 ~g of RNase A; lane 5, reverse transcription with 2.5 units of T. aquaticus DNA polymerase in 20/zl of PCR buffer with 1 ~g of primer G, for 30 min at 68°. The sample was then treated to PCR amplification as for lane 3. Lane 6, direct PCR amplification with 2.5 units of T. aquaticus DNA polymerase, 1/~g each of primers G and F in 50/zl PCR buffer. Lanes 1 and 2 were plasmid cDNA and genomic DNA clones ( - 5 0 ng each), respectively, amplified directly. Lanes M are size markers of an equimolar mixture of TaqI- and Sau3AI-
[31]
REVERSETRANSCRIPTIONOF mRNA BY Taq DNA POLYMERASE
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2. Ten microliters of the c D N A mixture from step I a b o v e is amplified by PCR. The buffer is adjusted to 50 m M KC1, 2 m M MgCI2, 10 m M TrisHCI (pH 8.3) (1 x PCR buffer), 200 tzM for each dNTP. One m i c r o g r a m of both forward and reverse oligodeoxynucleotide primers and 2.5 units of AmpliTaq are added, and the final volume adjusted to 50/zl with water. Mineral oil (50 tzl) is overlaid on top of the reaction mix to prevent evaporation during the PCR cycles. 3. The reaction mixture is subjected to amplification for 30-35 cycles. The actual p a r a m e t e r s will vary depending on the length of the target sequence and the GC content of the primers. The values used in the examples shown here were as follow: 94 °, 1 min; 55 °, 1 min; 72 °, 4 min, for 30 cycles using a Perkin-Elmer Cetus D N A thermal cycler (Fig. 1) and 94 °, 30 sec; 55 °, 30 sec; 72 °, 2 min, for 30-35 cycles using an M J Research (Watertown, MA) p r o g r a m m a b l e thermal controller (Fig. 2). The last cycle includes an incubation at 72 ° for an extra 5 min. 4. After completion of the PCR cycles, the mineral oil is carefully extracted twice with 500/zl of chloroform and the aqueous phase stored at - 20 °. Eight microliters of the reaction mix is analyzed by agarose gel electrophoresis. Fifty-milliliter agarose gels (1.4-1.6% agarose) in 1 x T B E buffer, containing 50/zg/ml ethidium bromide, are run submarine fashion at 60-mA constant current. The PCR-amplified D N A fragments can be analyzed by standard Southern blot analysis 8 using cloned plasmid sequences containing the PCR target sequences. Discussion The experiments shown were designed to reverse transcribe m R N A into c D N A with primers specific for the human glucose-6-phosphate dehydrogenase gene (G6PD) 4'5 and a conserved viral gene from human herpesvirus 6 (HHV-6). 9 The primers were chosen in each case to cross an intron such that amplification from m R N A could be distinguished from genomic D N A . R e v e r s e transcription of G6PD-specific R N A with either A M V 8 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Press, Cold Spring Harbor, New York, 1982. 9 G. L. Lawrence, M. Chee, M. A. Craxton, U. A. Gompels, R. W. Honess, and B. G. Barrell, J. Virol. 64, 287 (1990).
digested pBS. (A) Ethidium bromide-stained agarose gel; (B) and (C) are two different exposures of the Southern blot probed with the plasmid cDNA clone. Primers G and F amplify a genomic DNA fragment of 657 bp and an mRNA fragment of 358 bp. (Reproduced from Jones and Foulkes4 with permission of Oxford University Press.)
418
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189 -105
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FIG. 2. HHV-6 reverse transcription by AMV reverse transcriptase and T. aquaticus DNA polymerase. Aliquots of total cytoplasmic RNA from HHV-6-infected HSB-2 cells, -2/~g, were reverse transcribed in 1 x PCR buffer (final volume, 20 t~l) with 50 ng primer 6SPI, 40 units AMV reverse transcriptase at 42° for 60 min (lanes 1-3) or 2.5 units of AmpliTaq at 72° for 60 min (lanes 4-6). Samples (10/~1) were then submitted to amplification in 1 × PCR buffer (50-~1 final volume) with 0.5/~g each of primers 6SPI and 6SP2, and 2.5 units of AmpliTaq. Samples were amplified through 30 cycles (lanes 1-3) or 35 cycles (lanes 4-6). In lanes 2 and 5 the RNA was incubated with 10 ~g of RNase A for 60 min at 37° prior to reverse transcription, and lanes 3 and 6 were pretreated with 10 units of RNase-free DNase for 60 rain at 37°. Lanes M are size markers of an equimolar mixture of TaqI- and Sau3AIdigested pBluescript KS( - ). The arrowed genomic PCR fragment is 3623 bp in size and the cDNA fragment is 426 bp.
r e v e r s e t r a n s c r i p t a s e o r T. aquaticus D N A p o l y m e r a s e r e s u l t e d a f t e r P C R a m p l i f i c a t i o n in a s m e a r o f D N A f r a g m e n t s (Fig. 1A). S p e c i f i c a m p l i f i c a t i o n o f t h e G 6 P D c D N A s e q u e n c e s w a s c o n f i r m e d b y S o u t h e r n b l o t t i n g (Fig. 1B a n d C). N o c o n t a m i n a t i n g n u c l e a r g e n o m i c D N A w a s d e t e c t e d , as s h o w n b y t h e a b s e n c e o f a D N A b a n d o f 657 bp. R N a s e d i g e s t i o n p r i o r to P C R c o m p l e t e l y d e s t r o y e d a n y G 6 P D c D N A a m p l i f i c a t i o n (Fig. 1, l a n e 4). R e v e r s e t r a n s c r i p t i o n b y T. aquaticus D N A p o l y m e r a s e is c a p a b l e o f g i v i n g r i s e to l a r g e f r a g m e n t s , as s o m e o f t h e a r t i f a c t u a l b a n d s g e n e r a t e d a r e g r e a t e r t h a n 1000 b p in size (Fig. 1A, l a n e 5). H u m a n h e r p e s v i r u s 6 i n f e c t i o n o f cells l e a d s to v i r a l p a r t i c l e s in t h e cytoplasm, and preparation of cytoplasmic RNA concomitantly isolates v i r a l g e n o m i c D N A . T h e i n t r o n p r e s e n t in t h e g e n e is o v e r 3000 b p in size.9 T h i s " c o n t a m i n a t i n g " D N A w a s c o n c l u s i v e l y s e e n in t h e R N A s a m p l e and was successfully eliminated by pretreatment of the RNA with DNase
[31]
REVERSETRANSCRIPTIONOF mRNA BY Taq DNA POLYMERASE
419
(Fig. 2). This in effect released primers sequestered by genomic DNA, which could be utilized to amplify c D N A (Fig. 2, compare lanes 1 and 3, and 4 and 6). The correct c D N A band was confirmed by Southern blot analysis with an H H V - 6 recombinant plasmid clone (data not shown). Thus, if contaminating D N A is present in an RNA sample, DNase digestion will improve the yield of PCR-amplified cDNA. The efficiency of reverse transcription by T. aquaticus DNA polymerase is low compared with AMV reverse transcriptase, approximately - 1 % . This is not surprising, as T. aquaticus DNA polymerase has evolved to utilize a D N A template rather than a RNA one. A simple way to o v e r c o m e the inefficient reverse transcription step is to perform the PCR amplification for 40 or more cycles. The gene for T. aquaticus DNA polymerase has been cloned and expressed in E. coli, I° and with sitedirected mutagenesis it should be possible to engineer an enzyme with an improved reverse transcriptase activity. Analysis of the specificities of other thermostable D N A polymerases, for example, the enzyme isolated from Thermus thermophilus, may reveal polymerases with better reverse transcriptase activity.ll A requirement for 2-3 mM MgC12 was found for the reverse transcription of the G6PD sequences by T. aquaticus DNA polymerase. 4 Thus, as with PCR, care must be exercised in the magnesium ion concentration in the reaction buffer. Investigation into the replacement of magnesium with manganese may improve the reverse transcriptase activity of T. aquaticus D N A polymerase. 11 Tse and Forget 12 have also shown the ability of T. aquaticus D N A polymerase to carry out reverse transcriptions. They obtained reverse transcription by directly amplifying the RNA under standard PCR conditions, without a transcription preincubation step. We have not been able to amplify R N A directly by PCR (Fig. 1, lane 6), 4 but this may be a reflection of the actual R N A sequences involved. In conclusion, T. aquaticus D N A polymerase has been shown to possess reverse transcriptase activity and this e n z y m e (or engineered variants and other thermostable polymerases) should prove extremely useful for the analysis of m R N A structure. Acknowledgments The work described was supported by grants from the WellcomeTrust, Cancer Research Campaign, the Nuffield Foundation, and the Society for General Microbiology. l0 F. C. Lawyer, S. Stoffel, R. K. Saiki, K. Myambo, R. Drummond, and D. H. Gelfand, J. Biol. Chem. 264, 6427 (1989). ii Perkin-Elmer Cetus is marketing T. thermophilus DNA polymerase as a single enzyme for both reverse transcription and PCR amplification. t2 W. T. Tse and B. G. Forget, Gene 88, 293 (1990).