Abstract
Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.
Footnotes
PCR, polymerase chain reactionRT, reverse transcription; PBS, phosphate-buffered saline; DTT, dithiothreitol; m.o.i., multiplicity of infection;
References
REFERENCES
- 1.Barnes W.M. Proc. Natl. Acad. Sci. USA. 1994;91:2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Cheng S., Fockler C., Barnes W.M., Higuchi R. Proc. Natl. Acad. Sci. USA. 1994;91:5695–5699. doi: 10.1073/pnas.91.12.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cheng S., Chang S.Y., Gravitt P., Respess R. Nature. 1994;369:684–685. doi: 10.1038/369684a0. [DOI] [PubMed] [Google Scholar]
- 4.Cheng S., Higuchi R., Stoneking M. Nature Genet. 1994;7:350–351. doi: 10.1038/ng0794-350. [DOI] [PubMed] [Google Scholar]
- 5.Cheng S., Chen Y., Monforte J.A., Higuchi R., Van Houten B. PCR Methods Appl. 1995;4:294–298. doi: 10.1101/gr.4.5.294. [DOI] [PubMed] [Google Scholar]
- 6.Tellier R., Bukh J., Emerson S.U., Purcell R.H. Proc. Natl. Acad. Sci. USA. 1996;93:4370–4373. doi: 10.1073/pnas.93.9.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Martinez J.M., Breidenbach H.H., Cawthon R. Genome Res. 1996;6:58–66. doi: 10.1101/gr.6.1.58. [DOI] [PubMed] [Google Scholar]
- 8.Chumakov K.M. J. Virol. 1996;70:7331–7334. doi: 10.1128/jvi.70.10.7331-7334.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Fakhfakh H., Vilaine F., Makni M., Robaglia C. J. Gen. Virol. 1996;77:519–523. doi: 10.1099/0022-1317-77-3-519. [DOI] [PubMed] [Google Scholar]
- 10.Nathan M., Mertz L.M., Fox D.K. Focus. 1995;17:78–80. [Google Scholar]
- 11.Herold J., Raabe T., Siddell S. Arch. Virol. [Suppl.] 1993;7:63–74. doi: 10.1007/978-3-7091-9300-6_6. [DOI] [PubMed] [Google Scholar]
- 12.Raabe T., Schelle-Prinz B., Siddell S.G. J. Gen. Virol. 1990;71:1065–1073. doi: 10.1099/0022-1317-71-5-1065. [DOI] [PubMed] [Google Scholar]
- 13.Siddell S. J. Gen. Virol. 1983;64:113–125. doi: 10.1099/0022-1317-64-1-113. [DOI] [PubMed] [Google Scholar]
- 14.Meinkoth J., Wahl G. Anal. Biochem. 1984;138:267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
- 15.Gerard G.F., Schmidt B.J., Kotewicz M.L., Campbell J.H. Focus. 1992;14:91–93. [Google Scholar]
- 16.Koenig M., Hoffmann E.P., Bertelson C.J., Monaco A.P., Feener C., Kunkel L.M. Cell. 1987;50:509–517. doi: 10.1016/0092-8674(87)90504-6. [DOI] [PubMed] [Google Scholar]
- 17.Hoffman E.P., Monaco A.P., Feener C.C., Kunkel L.M. Science. 1987;238:347–350. doi: 10.1126/science.3659917. [DOI] [PubMed] [Google Scholar]
- 18.Tennyson C.N., Shi Q., Worton R.G. Nucleic Acids Res. 1996;24:3059–3064. doi: 10.1093/nar/24.15.3059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Racaniello V.R., Baltimore D. Science. 1981;214:916–919. doi: 10.1126/science.6272391. [DOI] [PubMed] [Google Scholar]
- 20.Rice C.M., Levis R., Strauss J.H., Huang H.V. J. Virol. 1987;61:3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rice C.M., Grakoui A., Galler R., Chambers T.J. New Biol. 1989;1:285–296. [PubMed] [Google Scholar]
- 22.Sumiyoshi H., Hoke C.H., Trent D.W. J. Virol. 1992;66:5425–5431. doi: 10.1128/jvi.66.9.5425-5431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]