Skip to main content
SpringerLink
Log in
Book cover

PCR Cloning Protocols pp 37–51Cite as

XL PCR Amplification of Long Targets from Genomic DNA

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 192))

Abstract

Long polymerase chain reaction (PCR)>(1 5), specifically, XL PCR (Extra-Long Polymerase Chain Reaction), has enabled amplification of expanded trinucleotide repeats of the neuromuscular disease myotonic dystrophy (6) a 9-kb HIV-1 provirus from primary isolate DNA (7), 24.2-kb fragments from nanogram quantities of genomic DNA for DNA damage repair (8), and up to 42 kb of human genomic DNA (4). The capability of the long PCR process stems through the use of:

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   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

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. Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template integrity is essential for PCR amplification of 20-to 30-kb sequences from genomic DNA.PCR Meth. Appli. 4, 294–298.

    CAS  Google Scholar 

  2. Cheng, S. (1995) Longer PCR amplifications, in PCR Strategies (Innis M. A., Gelfand, D. H., and Sninsky, J. J., eds.) Academic, San Diego, CA, pp. 313–324.

    Google Scholar 

  3. Cheng, S., Chang, S.-Y., Gravitt, P., and Respess, R. (1994) Long PCR. Nature 369, 684–685.

    Article  CAS  PubMed  Google Scholar 

  4. Cheng, S., Fockler, 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  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Brugnoni, R., Morandi, L., Brambati, B., Briscioli, V., Cornelio, F., and Mantegazza, R. (1998) A new non-radioactive method for the screening and prenatal diagnosi of myotonic dystrophy patients. J. Neurol. 245, 289–293.

    Article  CAS  PubMed  Google Scholar 

  7. Salminen, M. O., Koch, C., Sanders-Buell, E., Ehrenberg, P. K., Michael, N. L., Carr, J. K., Burke, D. S., and McCutchan, F. E. (1995) Recovery of virtrually full length HIV-1 provirus of Diverse Subtypes from primary virus cultures using the polymerase chain reaction. Virology 213, 80–86.

    Article  CAS  PubMed  Google Scholar 

  8. Van Houten, B., Cheng, S., and Chen, Y. (2000) Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nonogram quantities of DNA. Mutation Research 460, 81–94.

    Article  PubMed  Google Scholar 

  9. Landre, P. A., Gelfand, D. H., and Watson, R. M. (1995) The use of cosolvents to enhance amplification by the polymerase chain reaction, in PCR Strategies (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., eds.) Academic, San Diego, CA, pp.3–16.

    Chapter  Google Scholar 

  10. Skera, A. (1992) Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerase with proofreading activity. Nucl. Acids Res. 20, 3551–3554.

    Article  Google Scholar 

  11. de Noronha, C. and Mullins, J. (1992) PCR Meth. Appli. 2, 131–136.

    Google Scholar 

  12. Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucl. Acids Res. 16, 1215.

    Article  CAS  PubMed  Google Scholar 

  13. Sambrook, J., Fitsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 6.20, 6.21, 9.16-9.19, 9.34-9.57, and B.23-B.24.

    Google Scholar 

  14. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols (Innis,M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., ed.s) Academic, San Diego, CA, pp.3–12.

    Google Scholar 

  15. Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. (1991) The effect of temperature and oligonucleotide primer length on the specificaity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol. 10, 233–238.

    Article  CAS  PubMed  Google Scholar 

  16. Schmid, C. W. and Jelinek, W. R. (1982) The Alu family of dispersed repetitive sequences.Sciences 216, 1065–1070.

    Article  CAS  Google Scholar 

  17. Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Block, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.Nucl. Acids Res. 20, 1717–1723.

    Article  CAS  PubMed  Google Scholar 

  18. Monforte, J. A., Winegar, R. A., and Rudd, C. J. (1994) Megabase genomic DNA isolation procedure for use in transgenic mutagenesis assays. Environ. Mol. Mutagen. 23, 46.

    Google Scholar 

  19. Mullenbach, R., Lagoda, P. J. L., and Welter, C. (1989) Technical Tips: an efficient salt-chloroform extraction of DNA from blood and tissues. Trends in Gen. 5, 391.

    CAS  Google Scholar 

  20. Kolmodin, L., Cheng, S., and Akers, J. (1995) GeneAmp XL PCR Kit, in Amplifications:A Forum for PCR Users (The Perkin Elmer Corporation), Issue 13.

    Google Scholar 

  21. Cheng, S., Higuchi, R., and Stoneking, M. (1994) Complete mitochondrial genome amplification.Nature Gen. 7, 350, 351.

    Article  CAS  Google Scholar 

  22. Robin, E. D. and Wong, R. (1988) Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell Physiol. 136, 507–513.

    Article  CAS  PubMed  Google Scholar 

  23. Jurecic, R., Nachtman, R. G., Colicos, S. M., and Belmont, J. W. (1998) Identification and Cloning of Differentially Expressed Genes by Long-Distance Differential Display. Anal.Biochem. 259, 235–244.

    Article  CAS  PubMed  Google Scholar 

  24. Scharf, S. J., (1990) Cloning with PCR, in PCR Protocols (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.) Academic, San Diego, CA, pp. 84–91.

    Google Scholar 

  25. Fromenty, B., Demeilliers, Mansouri, A., and Pessayre, D. (2000) Escherichia coli exonu-clease III enhances long PCR amplification of damaged DNA templates. Nucl. Acids Res. 28, 50.

    Article  Google Scholar 

  26. Carle, G. F., Frank, M., and Olson, M. V. (1989) Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232, 65–68.

    Article  Google Scholar 

  27. Hengen, P. N. (1997) Methods and reagents: optimizing multiplex and LA-PCR with betaine. TIBS 22, 225, 2226.

    CAS  PubMed  Google Scholar 

  28. Clark, J. M. (1988) Novel nontemplated nucleotide addition reactions catalyzed by prokaryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16, 9677–9686.

    Article  CAS  PubMed  Google Scholar 

  29. Hu, G. (1993) DNA Polymerase-catalyzed addition of nontemplated extra nucleotides to the 3′end of a DNA fragment. DNA Cell Biol. 12, 763–770.

    Article  CAS  PubMed  Google Scholar 

  30. Stewart, A. C., Gravitt, P. E., Cheng, S., and Wheeler, C. M. (1995) Generation of entire human papilloma virus genomes by long PCR: frequency of errors produced during amplification. Genome Res. 5, 79–88.

    Article  CAS  PubMed  Google Scholar 

  31. Costa, G. L. and Weiner, M. P. (1994) Protocols for cloning and analysis of blunt-ended PCR-generated DNA fragments. PCR Meth. Appl. 3, S95–S106.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Roche Molecular Systems, Pleasanton, CA

    Lori A. Kolmodin

Authors
  1. Lori A. Kolmodin
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Plant Science, Rutgers University, New Brunswick, NJ

    Bing-Yuan Chen  & Harry W. Janes  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Kolmodin, L.A. (2002). XL PCR Amplification of Long Targets from Genomic DNA. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:037

Download citation

  • DOI: https://doi.org/10.1385/1-59259-177-9:037

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-969-8

  • Online ISBN: 978-1-59259-177-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation