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Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 16))

Abstract

Conventional polymerase chain reaction (PCR) enables reliable amplification of 3–4 kb of DNA (1) while attempts at optimisation has enabled 15.6 kb of λ DNA to be amplified (2). The maximum amplifiable length of PCR is limited by the low fidelity of the Thermus aquaticus (Taq) DNA polymerase (3), the most commonly used thermostable polymerase. It is believed that inadvertent nucleotide misincorporations during the PCR extension steps cause chain terminations (3). The Taq polymerase lacks proofreading properties (4) and thus is unable to correct such misincorporations. The higher extension KM value for a misincorporated nucleotide is thought to cause detachment of the Taq polymerase from template DNA.

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References

  1. Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent advances in the polymerase chain reaction Science 252, 1643–1651.

    Article  PubMed  CAS  Google Scholar 

  2. Kainz, P., Schmiedlechner, A., and Strack, H. B. (1992) In vitro amplification of DNA fragments greater than 10 kb. Anal. Btochem. 202, 46–49.

    Article  CAS  Google Scholar 

  3. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA. Proc. Natl. Acad Sci. USA 85, 9436–9440.

    Article  PubMed  CAS  Google Scholar 

  4. Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Bzochemistry 27, 6008–6013.

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  6. 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  PubMed  CAS  Google Scholar 

  7. Agostini, H T and Stoner, G. L. (1995) Amplification of the complete polyomavirus JC genome from brain, cerebrospinal fluid and urine using pre-PCR restriction enzyme digestion. J. Neurovirol. 1, 316–320.

    Article  PubMed  CAS  Google Scholar 

  8. Li, Y. Y., Hengstenberg, C., and Maisch, B. (1995) Whole mitochondrial genome amplification reveals basal level multiple deletions in mtDNA of patients with dilated cardiomyopathy. Biochem. Biophys. Res. Comm. 210, 211–218.

    Article  PubMed  CAS  Google Scholar 

  9. Reynier, P., Pellissier, J F., Harle, J. R, and Malthiery, Y. (1994) Multiple deletions of the mitochondrial DNA in polymyalgia rheumatica. Biochem. Biophys Res Comm 205, 375–380

    Article  PubMed  CAS  Google Scholar 

  10. Campuzano, V., Montermini, L., Molto, M. D., Pianese, L., Cossee, M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A., Zara, F., Canizares, J., Koutnikova, H., Bidichandani, S. I., Gellera, C., Brice, A., Trouillas, P., Demichele, G., Filla, A, Defrutos, R., Palau, F., Patel, P. I., Didonato, S., Mandel, J. L., Cocozza, S., Koenig, M., and Pandolfo, M. (1996) Friedreichs ataxia—an autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423–1427

    Article  PubMed  CAS  Google Scholar 

  11. Cheng, S., Barcelo, J. M., and Korneluk, R. G(1996) Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR. Hum Mutat 7, 304–310

    Article  PubMed  CAS  Google Scholar 

  12. Tellier, R., Bukh, J, Emerson, S. U, and Purcell, R H (1996) Amplification of the full-length hepatitis A virus genome by long reverse transcription PCR and transcription of infectious RNA directly from the amplicon Proc Natl Acad Sci USA 93, 4370–4373

    Article  PubMed  CAS  Google Scholar 

  13. Diachenko, L. B, Ledesma, J:, Chenchik, A. A., and Slebert, P. D (1996) Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed messenger RNA. Biochem. Biophys Res Comm. 219, 824–828.

    Article  PubMed  CAS  Google Scholar 

  14. Waggott, W., Lo, Y M D, Bastard, C, Gatter, K. C, Leroux, D, Mason, D Y, Boultwood, J., and Wainscoat, J. S. (1995) Detection of NPM-ALK DNA rearrangement in CD30 positive anaplastic large-cell lymphoma. Br J Haematol 89, 905–907

    Article  PubMed  CAS  Google Scholar 

  15. Sarris, A. H., Luthra, R, Papadimitracopoulou, V, Waasdorp, M, Dimopoulos, M A, McBride, J A., Cabanillas, F., Duvic, M, Deuseroth, A, Morris, S. W, and Pugh W. C. (1996) Amplification of genomic DNA demonstrates the presence of the t(2-5)(p23-q35) in anaplastic large-cell lymphoma, but not in other non-Hodgkins lymphomas, Hodgkins disease, or lymphomatoid papulosis. Blood 88, 177l–1779

    Google Scholar 

  16. Akasaka, T., Muramatsu, M, Ohno, H., Miura, I, Tatsumi, E., Fukuhara, S., Mori, T., and Okuma, M. (1996) Application of long distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B-cell neoplasms Blood 88, 985–994.

    PubMed  CAS  Google Scholar 

  17. Morris, S. W., Kirstein, M. N., Valentine, M. B., Dittmer, K. G., Shapiro, D. N, Saltman, D. L., and Look, A. T. (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkins lymphoma. Science 263, 1281–1284.

    Article  PubMed  CAS  Google Scholar 

  18. Gunther, S., Li, B C, Miska, S., Kruger, D H., Meisel, H, and Will, H (1995) A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol. 69, 5437–5444.

    PubMed  CAS  Google Scholar 

  19. Maclaren, D C and Clarke, S (1996) Rapid mapping of genomic pl clones—the mouse L-isoaspartyl/D-aspartyl methyltransferase gene. Genomics 35, 299–307

    Article  PubMed  CAS  Google Scholar 

  20. Machlda, M., Manabe, M, Yasukawa, M., and Jigami, Y (1996) Applicaition of long-distance PCR to restriction site mapping of a cloned DNA fragment on the lambda-EMBL3 phage vector Biosci Biotechnol. Biochem 60, 101l–l013

    Google Scholar 

  21. Li, D. Z. and Vijg, J (1996) Multiplex coamplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR Nucleic Acids Res 24, 538,539

    Article  PubMed  CAS  Google Scholar 

  22. Cline, J., Braman, J.C., and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24, 3546–3551.

    Article  PubMed  CAS  Google Scholar 

  23. Cheng, S, Chen, Y M, Monforte, J. A., Higuchi, R, and Vanhouten, B. (1995) Template integrity is essential for PCR amplification of 20 kb to 30 kb sequences from genomic DNA. PCR Methods Appl 4, 294–298

    PubMed  CAS  Google Scholar 

  24. Melov, S., Lithgow, G. J, Fischer, D. R., Tedesco, P M., and Johnson, T E. (1995) Increased frequency of deletions in the mitochondrial genome with age of Caenorhabditis elegans. Nucleic Acids Res 23, 1419–1425.

    Article  PubMed  CAS  Google Scholar 

  25. Ermak, G., Jennings, T., Robinson, L., Ross, J. S., and Figge, J. (1996) Restricted patterns of CD44 variant exon expression in human papillary thyroid carcinoma Cancer Res 56, 1037–1042.

    PubMed  CAS  Google Scholar 

  26. Stewart, A., Gravitt, P. E, Cheng, S., and Wheeler, C M. (1995) Generation of entire human papillomavirus genomes by long PCR-frequency of errors produced during amplification PCR Methods Appl. 5, 79–88.

    CAS  Google Scholar 

  27. Ling, M. F. and Robinson, B. H (1995) A one-step polymerase chain reaction site-directed mutagenesis method for large gene cassettes with high efficiency, yield, and fidelity Anal Biochem 230, 167–172.

    Article  PubMed  CAS  Google Scholar 

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© 1998 Humana Press Inc., Totowa, NJ

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Waggott, W. (1998). Long Range PCR. In: Lo, Y.M.D. (eds) Clinical Applications of PCR. Methods in Molecular Medicine™, vol 16. Humana Press. https://doi.org/10.1385/0-89603-499-2:81

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  • DOI: https://doi.org/10.1385/0-89603-499-2:81

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-499-0

  • Online ISBN: 978-1-59259-600-3

  • eBook Packages: Springer Protocols

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