Advertisement

Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction

  • Richard A. Gibbs
  • Phi-Nga Nguyen
  • C. Thomas Caskey
Part of the Methods in Molecular Biology book series (MIMB, volume 9)

Abstract

Protocols for the sequence analysis of conventional single-stranded or double-stranded DNA templates are often unsuitable for the direct sequencing of DNA fragments generated by the polymerase chain reaction (PCR) (1,2). The features that can distinguish PCR products as templates for sequencing include (a) contamination of the reactions by nonspecific PCR amplification products that are complementary to the sequencing primer, (b) the persistence of “leftover” PCR primers from the amplification reactions, and (c) the potential for competition between one strand of the amplified fragment and the oligonucleotide used for the sequencing. The various approaches that have been used to overcome these problems include
  1. 1.

    The use of 5′-end-labeled DNA-sequencing primers that are complementary to regions between the PCR primers (3);

     
  2. 2.

    Gel purification of amplified DNA to remove unwanted fragments and primer (4);

     
  3. 3.

    Spin columns for the separation of leftover primers from high mol wt material (5, 6);

     
  4. 4.

    “Asymmetric” or “unbalanced” PCR priming to generate an excess of single strands during the initial amplification (7);

     
  5. 5.

    Addition of dimethylsulfoxide (DMSO) to sequencing reactions with short annealing times (8); and

     
  6. 6.

    The use of several short, high-temperature, sequencing cycles (9).

     

Keywords

Polymerase Chain Reaction Single Strand Polymerase Chain Reaction Primer Polymerase Chain Reaction Buffer Short Annealing Time 
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.
    Mullis, K. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymnerase catalyzed chain reaction. Methods Enzymol 155, 335–350.PubMedCrossRefGoogle Scholar
  2. 2.
    Saiki, R. K., Scharf, F., Faloona, F., Mullis, K. B., Horn, G., Erlich, H. A., and Arnheim, N. (1985) Enzymatic amplification of β-globin gcnomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.PubMedCrossRefGoogle Scholar
  3. 3.
    Wrischnik, L. A., Higucht, R. C., Stoneking, M., Erlich, H. A., Arnheim, N., and Wilson, A. C. (1987) Length mutations in human mitochondrial DNA: Direct sequencing of enzymatic ally amplified DNA. Nucleic Acids Res. 15, 529–512.PubMedCrossRefGoogle Scholar
  4. 4.
    McMahon, G., Davis, E., and Wogan, G. N. (1988) Characterization of c-ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors. Proc. Natl. Acad. Sci. USA 84, 4974–4978.CrossRefGoogle Scholar
  5. 5.
    Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, R. G., Erlich, H. A., and Kazazian, H. H. Jr. (1987) Characterization of beta-thaiassemia mutations using direct gcnomic sequencing of amplified single copy DNA. Nature 30, 384–386.CrossRefGoogle Scholar
  6. 6.
    Yandell, D. W. (1989) Direct genomic sequencing of alleles at the retinoblastoma locus’ Applications to carrier diagnosis and genetic counselling, in Cancer Cells: Molecular Diagnostics of Human Cancer, vol 7, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 223–227.Google Scholar
  7. 7.
    Gyllensten, U. B. and Erlich, H. (1988) Generation of single stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus Proc Natl. Acad Sci. USA 85, 7652–7656.PubMedCrossRefGoogle Scholar
  8. 8.
    Winship, P. R. (1989) An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 17, 1266PubMedCrossRefGoogle Scholar
  9. 9.
    Carothers, A. M., Urlaub, G., Mucha, J., Grunberger, D., and Chasm, L. A. (1989) Point mutation analysis in a human gene: Rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method Biotechnuiques 7, 494–499Google Scholar
  10. 10.
    Kogan, S. C., Doherty, M., and Gitschier, J. (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences Application to hemophilia A. N. Engl. J. Med. 317, 985–990PubMedCrossRefGoogle Scholar
  11. 11.
    Gibbs, R. A., Chamberlain, J. S., and Caskey C. T. (1989) Diagnosis of new mutation diseases using the polymerase chain reaction, in The Polymerase Cham Reaction Principles and Applications (Erlich, H., ed.), Stockton, New York, pp. 171–191Google Scholar
  12. 12.
    Kawasaki, E. (1989) Detection of gene expression, in The Polymerase Chain Reaction. Principles and Applications (Erlich, H., ed.), Stockton, New York, pp. 89–97Google Scholar
  13. 13.
    Chirgwin, J. M., Przybyla, A. E., McDonald, R. J., and Rutter, W J (1979) Isolation of biologically active nbonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299.PubMedCrossRefGoogle Scholar
  14. 14.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S J, Higuchi, R, Horn, G T., and Mulhs, K. B. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  15. 15.
    Gibbs, R. A., Nguyen, P. N., McBride, L J, Koepf, S. M, and Caskey, C T (1989) Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of m vitro amplified cDNA Proc NatL Acad Set USA 89, 1919–1923.CrossRefGoogle Scholar
  16. 16.
    Innis, M A, Myambo, K. B, Gelfand, D. H., and Brow, M A (1988) DNA sequencing with Thermits acquaticus DNA polymerase and direct sequencing of polymerase chain reacuon amplified DNA Proc. Natl. Acad. Sci. USA 85, 9436–9440PubMedCrossRefGoogle Scholar
  17. 17.
    McBride, L J., Koepf, S. M, Gibbs, R A, Nguyen, P N, Salser, W, Mayrand, P E, Hunkapiller, M. W., and Kronick, M. N. (1989) Automated DNA sequencing methods using polymerase chain reacuon Clin. Chem, 35, 2196–2201PubMedGoogle Scholar
  18. 18.
    Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C, Connell, C. R., Heiner, C, Kent, S. B H., and Hood, L. E. (1986) Fluorescence detection in automated DNA sequence analysis Nature 321, 674PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press Inc., Clifton, NJ 1991

Authors and Affiliations

  • Richard A. Gibbs
    • 1
  • Phi-Nga Nguyen
    • 2
  • C. Thomas Caskey
    • 1
    • 2
  1. 1.Institute for Molecular GeneticsBaylor College of MedicineHouston
  2. 2.Howard Hughes Medical InstituteBaylor College of MedicineHouston

Personalised recommendations