Skip to main content
SpringerLink
Log in
Book cover

PCR Protocols pp 173–177Cite as

Long PCR Methodology

  • Protocol

  • 7074 Accesses

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 226))

Abstract

In this chapter, we detail protocols of long polymerase chain reaction (PCR) and long RT-PCR, which we have found to be versatile, sensitive, and straightforward to optimize. We have used these protocols with success on several different templates, including lambda phage DNA, HAV, HBV, HCV (1), torovirus (2), coxsackie B6 virus (3), and human beta galactosidase mRNA (R. Tellier, unpublished data). The guanine-cytosine (GC) content of these templates varied from 37.8 to 58.8%. These protocols have also been used on genomic human DNA with success (4).

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   74.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Tellier, R., Bukh, J., Emerson, S. U., Miller, R. H., and Purcell, R. H. (1996) Long PCR and its application to hepatitis viruses: Amplification of hepatitis A, hepatitis B and hepatitis C virus genomes. J. Clin. Microbiol. 34, 3085–3091. (Erratum published in J. Clin. Microbiol. 1997;35: 2713).

    PubMed  CAS  Google Scholar 

  2. Duckmanton, L. M., Tellier, R., Liu, P., and Petric, M. (1998) Bovine torovirus: Sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus. Virus Res. 58, 83–96.

    Article  PubMed  CAS  Google Scholar 

  3. Martino, T. A., Tellier, R., Petric, M., Irwin, D. M., Afshar, A., and Liu P. (1999) The complete consensus sequence of coxsackievirus B6 and generation of infectious clones by long RT-PCR. Virus Res. 64, 77–86.

    Article  PubMed  CAS  Google Scholar 

  4. Chen, B., Rigat, B., Curry, C, and Mahuran, D. J. (1999) Structure of the GM2A gene: identification of an exon 2 nonsense mutation and a naturally occurring transcript with an in-frame deletion of exon 2. Am. J. Hum. Genet. 65, 77–87.

    Article  PubMed  CAS  Google Scholar 

  5. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220.

    Article  PubMed  CAS  Google Scholar 

  6. Lawyer, F. C, Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D., et al. (1993) High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. Genome Res. 2, 275–287.

    Article  CAS  Google Scholar 

  7. Clontech (1998) User Manual for Advantage cDNA PCR kit and Advantage cDNA Polymerase Mix.

    Google Scholar 

  8. Cheng, S., Fockle, C., Barnes, W. M., and Higuchi, R. (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad Sci. USA 91 5695–5699.

    Article  PubMed  CAS  Google Scholar 

  9. Tellier, R., Bukh, J., Emerson, S. U., and Purcell, R. H. (1997) Amplification of the full length hepatitis A virus by long RT-PCR and generation of infectious RNA directly from the amplicon in Viral Hepatitis and Liver Disease, Proceedings of the IX Triennial International Symposium on Viral Hepatitis and Liver Disease (Rizzetto M., Purcell R. H., Gerin J. L., and Verme, G., eds.), Minerva Medica, Turin, pp. 48–50.

    Google Scholar 

  10. Gibco BRL (1994) Technical Bulletin 18064-2. Gibco BRL, Gaithersburg, MD.

    Google Scholar 

  11. Nathan, M., Mertz, L. M., and Fox, D. K. (1995) Optimizing long RT-PCR. Focus 17, 78–80.

    Google Scholar 

  12. Wang, L.-F., Radkowski, M., Vargas, H., Rakela, J., and Laskus, T. (1997) Amplification and fusion of long fragments of hepatitis C virus genome. J. Virol. Methods 68, 217–223.

    Article  PubMed  CAS  Google Scholar 

  13. Lindberg, A. M., Polacek, C., and Johansson, S. (1997) Amplification and cloning of complete enterovirus genomes by long distance PCR. J. Virol. Methods 65, 191–199.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Microbiology, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada

    Raymond Tellier

  2. Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD

    Jens Bukh, Suzanne U. Emerson & Robert H. Purcell

Authors
  1. Raymond Tellier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jens Bukh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzanne U. Emerson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert H. Purcell
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK

    John M. S. Bartlett

  2. Department of Hematology, Royal Infirmary of Edinburgh, Edinburgh, UK

    David Stirling

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc.

About this protocol

Cite this protocol

Tellier, R., Bukh, J., Emerson, S.U., Purcell, R.H. (2003). Long PCR Methodology. In: Bartlett, J.M.S., Stirling, D. (eds) PCR Protocols. Methods in Molecular Biology™, vol 226. Humana Press. https://doi.org/10.1385/1-59259-384-4:173

Download citation

  • DOI: https://doi.org/10.1385/1-59259-384-4:173

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-642-0

  • Online ISBN: 978-1-59259-384-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation