Advertisement

PCR Protocols pp 189-198 | Cite as

Identification of Alternatively Spliced mRNAs and Localization of 5' Ends by Polymerase Chain Reaction Amplification

  • Cheng-Ming Chiang
  • Louise T. Chow
  • Thomas R. Broker
Part of the Methods in Molecular Biology book series (MIMB, volume 15)

Abstract

Messenger RNAs of higher eucaryotes are usually modified posttran-scriptionally to contain 5′ caps and nucleotide methylation, 3′ polyadenylation, and one or more internal splices to remove introns and join exon segments into the mature protein-coding sequences. With the abilities of retroviral reverse transcriptases to create complementary DNA (cDNA) copies of RNA and of thermostable DNA polymerases to amplify specific DNA segments by repeated cycles of denaturation of duplex templates, annealing of complementary oligonucleotide primers, and strand elongation, it is becoming increasingly popular to use this highly sensitive polymerase chain reaction (PCR) to isolate partial cDNA sequences for the purpose of identifying RNA splice sites and inferring the coding capacity. The splice junction information from the partial cDNAs, together with additional biophysical or biochemical information, can then be employed to map the 5′ ends of the mRNAs and assign the AUG protein initiation codon and open reading frame to the message.

Keywords

Polymerase Chain Reaction Primer Splice Acceptor Site Sensitive Polymerase Chain Reaction Specific Polymerase Chain Reaction Primer Clone Polymerase Chain Reaction Product 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Rotenberg, M. O., Chow, L. T., and Broker, T. R. (1989) Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction. Virology 172, 489–497.PubMedCrossRefGoogle Scholar
  2. 2.
    Palermo-Dilts, D. A., Broker, T. R., and Chow, L. T. (1990) Human papillomavirus type 1 produces redundant as well as polycistronic mRNAs in plantar warts. J. Virol. 64, 3144–3149.PubMedGoogle Scholar
  3. 3.
    Chiang, C.-M., Broker, T. R., and Chow, L. T. (1991) An E1M E2C fusion protein encoded by human papillomavirus type 11 is a sequence-specific transcription repressor. J. Virol. 65, 3317–3329.PubMedGoogle Scholar
  4. 4.
    He, X., Treacy, M. N., Simmons, D. M., Ingraham, H. A., Swanson, L. W., and Rosenfeld, M. G. (1989) Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340, 35–42.PubMedCrossRefGoogle Scholar
  5. 5.
    Chang, T.-H., Arenas, J., and Abelson, J. (1990) Identification of five putative yeast RNA helicase genes. Proc. Natl. Acad. Sci. USA 87, 1571–1575.PubMedCrossRefGoogle Scholar
  6. 6.
    Hoey, T., Dynlacht, B. D., Peterson, M. G., Pugh, B. F., and Tjian, R. (1990) Isolation and characterization of the drosophila gene encoding the TATA box binding protein, TFIID. Cell 61, 1179–1186.PubMedCrossRefGoogle Scholar
  7. 7.
    Chow, L. T., Nasseri, M., Wolinsky, S. M., and Broker, T. R. (1987) Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata. J. Virol. 61, 2581–2588.PubMedGoogle Scholar
  8. 8.
    Chow, L. T., and Broker, T. R. (1989) Mapping the genetic organization of RNA by electron microscopy. Methods Enzymol. 180, 239–261.PubMedCrossRefGoogle Scholar
  9. 9.
    Berk, A. J. (1989) Characterization of RNA molecules by S1 nuclease analysis. Methods Enzymol. 180, 334–347.PubMedCrossRefGoogle Scholar
  10. 10.
    Muranyi, W., and Flugel, R. M. (1991) Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures. J. Virol. 65, 727–735.PubMedGoogle Scholar
  11. 11.
    Rotenerg, M. O., Chiang, C.-M., Ho, M. L., Broker, T. R., and Chow, L. T. (1989) Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer. Virology 172, 468–477.CrossRefGoogle Scholar
  12. 12.
    Meyerhans, A., Vartanian, J.-P., and Wain-Hobson, S. (1990) DNA recombination during PCR. Nucleic Acids Res. 18, 1687–1691.PubMedCrossRefGoogle Scholar
  13. 13.
    Mount, S. M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459–472.PubMedCrossRefGoogle Scholar
  14. 14.
    Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J. (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005.PubMedCrossRefGoogle Scholar
  15. 15.
    Nassai, M. and Rieger, A. (1990) PCR-based site-directed mutagenesis using primers with mismatched 3′-ends. Nucleic Acids Res. 18, 3077–3078.CrossRefGoogle Scholar
  16. 16.
    Rappolee, D. A. (1990) Optimizing the sensitivity of RT-PCR. Amplifications 4, 5–7.Google Scholar
  17. 17.
    MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. M. (1987) Isolation of RNA using guanidinium salts. Methods Enzymol. 152, 219–227.PubMedCrossRefGoogle Scholar
  18. 18.
    Kawasaki, E. (1989) Amplification of RNA sequences via complementary DNA (cDNA). Amplifications 3, 4–6.Google Scholar
  19. 19.
    Kawasaki, E. (1990) Amplification of RNA, in PCR Protocols (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, CA, pp. 21–27.Google Scholar
  20. 20.
    Mocharla, H., Mocharla, R., and Hodes, M. E. (1990) Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping. Gene 93, 271–275.PubMedCrossRefGoogle Scholar
  21. 21.
    Grillo, M., and Margolis, F. L. (1990) Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes. BioTechniques 9, 262–268.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1993

Authors and Affiliations

  • Cheng-Ming Chiang
    • 1
    • 2
  • Louise T. Chow
    • 1
  • Thomas R. Broker
    • 1
  1. 1.Department of BiochemistyUniversity of Rochester School of MedicineRochesterNY
  2. 2.Laboratory of Biochemistry and Molecular BiologyThe Rockefeller UniversityNew York

Personalised recommendations